Colloidal gold immune test strip for intraoperative instant detection of IDH1 mutant protein as well as preparation method and application of colloidal gold immune test strip

Abstract

The invention discloses a preparation method and application of a colloidal gold immunochromatography test strip for detecting IDH1 mutant protein, and belongs to the field of rapid immunodetection. The colloidal gold chromatography test strip comprises a bottom plate, a sample pad, a nitrocellulose membrane, a water absorption pad and colloidal gold, the nitrocellulose membrane is sequentially provided with a detection line T and a quality control line C in the flowing direction of a sample, the detection line T is coated with IDH1 mutant protein, and the quality control line C is coated with a second antibody corresponding to a monoclonal antibody of the IDH1 mutant protein. Clinical tumor tissue samples collected in real time in glioma resection surgery are treated, an object to be detected and an immune gold label are mixed, incubated and dropped in a test strip sample adding hole, and the object to be detected runs along the test strip under the driving of a buffer solution and generates visual color change. The test strip has the advantages of being rapid in reaction, easy and convenient to operate, convenient to carry, high in specificity and the like, makes up the blank of an IDH1 mutant protein concentration quantitative method, and has good clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay analysis, and in particular relates to a colloidal gold immunoassay paper for real-time detection of IDH1 mutant protein during surgery, a preparation method and an application thereof. Background Art

[0002] Glioma is the most common malignant tumor of the central nervous system in the brain, accounting for approximately 30% of all brain tumors and 80% of all malignant brain tumors. It is characterized by high recurrence rate, high disability and mortality rate, and low survival rate. Surgical resection with adjuvant chemotherapy is crucial to improving the prognosis of glioma patients. The 2021 World Health Organization Classification of Central Nervous System Tumors emphasizes the importance of isocitrate dehydrogenase 1 (IDH1) mutation status in the classification of gliomas. Normally expressed IDH1 converts isocitrate into α-ketoglutarate. When IDH1 mutates, α-ketoglutarate is converted into 2-hydroxyglutarate. Abnormal accumulation of 2-hydroxyglutarate affects the occurrence and development of gliomas by affecting mechanisms such as DNA methylation, histone methylation modification, and cellular energy metabolism. Related studies have shown that IDH1 mutant gliomas are associated with survival benefits in patients receiving chemotherapy, immunotherapy, and targeted therapy, while wild-type IDH1 is not associated. For example, the preferred chemotherapy regimen for glioma patients harboring IDH1 mutations is temozolomide plus adjuvant PCV (procarbazine, lomustine, and vincristine). The combination of an IDH1 R132H-specific vaccine and a programmed death protein 1 (PD-1) inhibitor reduces glioma size and prolongs survival. IDH1 mutation inhibitors such as ivosidenib target the mutation site and inhibit glioma growth. Therefore, determining IDH1 mutation status during surgical biopsy is crucial for selecting individualized subsequent treatment strategies.

[0003] In traditional brain glioma treatment, immunohistochemistry and gene sequencing are usually used for detection. However, immunohistochemistry is cumbersome to operate, and the dewaxing and staining steps are time-consuming, and the interpretation of the results is highly dependent on the pathologist; gene sequencing technology has high requirements for testing instruments, requires the inspectors to have skilled operation techniques, and is time-consuming. As a result, the test results obtained by this method cannot effectively guide subsequent surgical plans during surgery. Therefore, there is an urgent need for clinical detection methods that can quickly detect the IDH1 mutation status within a few minutes during surgery, that is, in the operating room. It is necessary to develop a highly sensitive, convenient and fast analytical method for the immediate detection of IDH1 mutations.

[0004] Colloidal gold competitive immunochromatographic test strips are a commonly used detection method, widely used in disease diagnosis and biotoxin detection. This technology offers the advantages of short response time, ease of use, and low cost. It also requires neither specialized operators nor extensive laboratory equipment, making it a significant advantage in emergency clinical testing and large-scale screening. Currently, immunocolloidal gold test strips have been used for rapid intraoperative testing of parathyroid glands and for assessing Cyfra21-1 levels in lymph nodes to determine the presence of thyroid cancer metastases. These test strips offer an effective method for rapid intraoperative detection of IDH1 mutation status in gliomas. However, quantitative determination of mutant IDH1 protein concentrations in actual samples has not yet been reported. Therefore, developing a rapid intraoperative test for IDH1 mutant protein to rapidly stratify glioma pathology and subtypes is crucial for personalized glioma treatment and clinical prognosis. Summary of the Invention

[0005] To solve the above problems, the present invention discloses a colloidal gold immunoassay paper for real-time detection of IDH1 mutant protein during surgery, as well as a preparation method and application thereof, which is used to guide the selection of surgical strategies for glioma resection and postoperative treatment plans.

[0006] The technical solutions of the present invention are as follows:

[0007] A colloidal gold competitive immunochromatographic test strip for detecting IDH1 mutant protein comprises a base plate, a sample pad, a nitrocellulose membrane, a water-absorbing pad and colloidal gold. The nitrocellulose membrane is provided with a detection T line coated with the IDH1 mutant protein and a quality control C line coated with a goat anti-mouse secondary antibody corresponding to the IDH1 mutant monoclonal antibody in sequence along the sample flow direction.

[0008] A method for preparing a colloidal gold competitive immunochromatographic test strip for detecting IDH1 mutant proteins comprises the following steps:

[0009] Step (1) Preparation of immunogold label: Use 0.1M potassium carbonate solution to adjust the pH of colloidal gold solution, the volume ratio of colloidal gold solution to potassium carbonate solution is 1000:2-10, and mix evenly; add IDH1 mutant monoclonal antibody, mix evenly, and react at 25-40°C for 0.5-1.5h; add casein solution (final concentration is 0.5%-1.5%) to block for 0.5-1.5h, centrifuge, discard the supernatant solution, add storage solution, and store in a refrigerator at 4°C; the storage solution composition is 5%-15% sucrose, 0.5%-2% casein phosphate buffer.

[0010] Step (2) attaching a sample pad, a nitrocellulose membrane, and a water-absorbing pad to the bottom plate in sequence, with overlapping portions of 2 mm each;

[0011] Step (3) using a membrane streaking instrument to streak the IDH1 mutant protein and goat anti-mouse secondary antibody onto a nitrocellulose membrane to form a detection T line and a quality control C line;

[0012] Step (4) cutting the base plate and attached materials into test strips with a width of 3-5 mm by a strip cutting machine;

[0013] Step (5) loading the test strip in step (4) into a cartridge, which has two windows, a sample loading area and a color development area, with the sample loading area aligned with the sample pad area and the color development area aligned with the T-line and C-line areas;

[0014] Step (6) The test strips are placed in an aluminum foil bag containing a desiccant and stored at 4°C.

[0015] Preferably, in step (1), the final concentration of the IDH1 mutant monoclonal antibody in the colloidal gold solution is 9 μg / mL.

[0016] Preferably, in step (2), in the colloidal gold competitive immunochromatographic test strip, the length of the nitrocellulose membrane is 2.5-3 cm, the length of the sample pad is 2-2.5 cm, and the length of the absorbent pad is 1.5-2 cm.

[0017] Preferably, in step (3), the concentration of the protein coated on the detection T-line is 0.6-3 mg / mL, and the protein spraying volume of the protein coated on the detection T-line is 0.5-2 μL / cm.

[0018] Preferably, in step (3), the protein concentration of the quality control C line coating is 0.3-2 mg / mL, and the protein spraying amount of the quality control C line coating is 0.5-2 μL / cm.

[0019] Preferably, in step (3), the distance between the detection T line and the quality control C line is 5-10 mm.

[0020] Preferably, the nitrocellulose membrane is a Sartorius nitrocellulose membrane CN140 membrane.

[0021] Furthermore, the cartridge of the colloidal gold competitive immunochromatographic test strip has a cartridge cavity for placing the colloidal gold test strip. Two windows, a sample loading area and a color development area, are provided on the cartridge. The sample loading area is aligned with the sample pad area, and the color development area is aligned with the T line and C line areas of the nitrocellulose membrane of the colloidal gold test strip.

[0022] During glioma surgery, glioma tissue is lysed. The lysed sample is mixed with the immunogold label and then applied to the sample pad. After 8 minutes, the results are observed visually or captured with a smartphone. Images are processed and analyzed using Image J software. The grayscale value (relative intensity) ratio of the T-line to the C-line is plotted as the vertical axis, and the concentration of the IDH1 mutant protein standard solution is plotted as the horizontal axis to create a linear equation. The test results are substituted into the linear equation to calculate the IDH1 mutant protein content in the sample, enabling rapid intraoperative detection of IDH1 mutation status.

[0023] Clinical tumor tissue samples collected immediately during glioma resection surgery are processed, the test substance is mixed with the immunogold label, and then dripped onto the sample well of the test strip. Driven by the buffer solution, the solution flows along the test strip, producing a visible color change. If the patient's glioma is IDH1 wild-type, both the T and C lines will be colored; if the patient's glioma is IDH1 mutant, only the C line will be colored.

[0024] Beneficial effects of the present invention:

[0025] 1. The present invention provides a colloidal gold competitive immunochromatographic test strip for detecting IDH1 mutant protein in brain glioma tissue lysate and a preparation method thereof. By optimizing the synthesis of the immunogold label and the composition of the storage solution, the probe detection performance is improved while being stable. At the same time, the NC membrane used in the test strip, the coated IDH1 mutant protein and the concentration of the secondary antibody and the amount of immunogold label used are optimized to improve the detection performance of the test strip. The test strip can be used for rapid typing detection of mutant / wild-type brain gliomas. It is only necessary to visually identify the T / C line signal of the test strip to distinguish between IDH1 mutant and IDH1 wild-type patients. Previously, there was no analytical method for quantitative detection of IDH1 mutant protein concentration and no intraoperative instant detection strategy.

[0026] 2. This test strip detects IDH1 mutation status during surgery, helping glioma surgeons adopt the most optimized resection strategy. For IDH1 mutant gliomas, a surgical plan is adopted to safely resect the total tumor volume that absorbs MRI contrast agents to the maximum extent possible. For IDH1 wild-type gliomas, a surgical plan is adopted to only resect areas with significantly enhanced absorption, thereby reducing the risk of neurological dysfunction caused by excessive resection of brain tissue. This also helps physicians adopt postoperative strategies that are related to the survival benefits of IDH1 mutant glioma patients and improve patient survival rates. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is an operational flow chart of the present invention;

[0028] Figure 2 is a UV-visible spectrum of colloidal gold obtained in an embodiment of the present invention;

[0029] Figure 3 is a transmission electron microscope image of colloidal gold in an embodiment of the present invention;

[0030] Figure 4 This is a diagram showing the optimal pH value determined by the salt precipitation experiment in an embodiment of the present invention;

[0031] Figure 5 This is a diagram of the optimal antibody determined by the salt precipitation experiment in an embodiment of the present invention;

[0032] Figure 6 is a particle size distribution diagram of immune colloidal gold according to an embodiment of the present invention;

[0033] Figure 7 This is a diagram of nitrocellulose membrane screening according to an embodiment of the present invention;

[0034] Figure 8 This is a T-line spraying amount optimization diagram of an embodiment of the present invention;

[0035] Figure 9 This is a diagram showing the optimized spraying amount of line C according to an embodiment of the present invention;

[0036] Figure 10 This is a diagram showing the optimization of the immunogold label dosage according to an embodiment of the present invention;

[0037] Figure 11 This is the test strip photography time optimization curve of an embodiment of the present invention;

[0038] Figure 12 This is a diagram of a standard sample test strip according to an embodiment of the present invention;

[0039] Figure 13 This is the pure sample standard curve of the test strip in the embodiment of the present invention;

[0040] Figure 14 1 is a graph showing the cross-reactivity rate of the test strips prepared in an embodiment of the present invention (from left to right: blank, IDH1 wild-type protein, carcinoembryonic antigen, cardiac troponin, prostate-specific antigen, and alpha-fetoprotein);

[0041] Figure 15 This is a diagram of the test strip of an embodiment of the present invention being tested in an actual sample;

[0042] Figure 16 This is a stability test diagram of a test strip according to an embodiment of the present invention. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. It should be noted that the terms "front," "rear," "left," "right," "up," and "down" used in the following description refer to directions in the accompanying drawings, and the terms "inward" and "outward" refer to directions toward or away from the geometric center of a particular component, respectively.

[0044] Example 1. Preparation of colloidal gold

[0045] (I) 100 mL of distilled water was placed in a three-necked flask and stirred in a constant temperature magnetic stirrer (300 rpm, 110°C). After the water was heated to boiling, 120 μL of 200 mM chloroauric acid was added and stirred evenly. 1 mL of 1% trisodium citrate solution was added and heated for 15 min until the solution changed from colorless to wine red. The solution was then stopped from heating and cooled to room temperature. The solution was concentrated by centrifugation to an absorbance of 1.2 and stored at 4°C in the dark.

[0046] (2) Colloidal gold was characterized by sensory evaluation, UV scanning and transmission electron microscopy.

[0047] (1) Sensory evaluation: Place the synthesized colloidal gold solution into a centrifuge tube and observe with the naked eye to preliminarily determine whether the colloidal gold solution is qualified (color, transmittance). Figure 1 As shown, the appearance is wine red, clear and transparent, with no precipitation.

[0048] (2) The colloidal gold solution was subjected to UV spectrophotometry to measure its absorption spectrum at 400-700 nm, determine the wavelength of its maximum absorption peak, and observe the peak shape and peak width of the maximum absorption peak. Figure 2 , the colloidal gold solution has a maximum absorption peak at 525nm; OD 525 nm =1.204; and the maximum absorption peak in the absorption spectrum is smooth and tall, indicating that the prepared colloidal gold particles are relatively uniform and have good dispersibility.

[0049] (3) Microstructure of colloidal gold: Dilute the colloidal gold solution 5 times with distilled water, take 8 μL of the colloidal gold solution and drop it on the surface of a 200-mesh carbon film copper grid, and vacuum dry it overnight. Under a transmission electron microscope, observe the size and morphology of the colloidal gold particles and determine their average particle size. Figure 3 As shown, the colloidal gold nanoparticles are uniform in size, well dispersed, and spherical with a particle size of approximately 20 nm. This result is consistent with that obtained by UV-Vis absorption spectroscopy. These results demonstrate the successful preparation of a colloidal gold solution with uniform particle size and good stability, preparing the next step for the preparation of immunogold labels.

[0050] Example 2. Preparation of immunogold label

[0051] (1) Determination of the labeled pH value.

[0052] The dosage of IDH1 mutant monoclonal antibody was 6 μg / mL, the antibody diluent was 10 mM phosphate buffer (pH 7.4), the reaction time was 1 h, the blocking time was 0.5 h, and the pH of the colloidal gold solution was adjusted by adding different amounts of 0.1 M potassium carbonate solution. The specific steps are as follows:

[0053] (1) Adjust the pH value: add 200 μL of colloidal gold solution to a centrifuge tube, add 0.1 M potassium carbonate solution from left to right in increasing volumes (Table 1), and vortex to mix;

[0054] Table 1

[0055] Test tube number 1 2 3 4 5 6 7 Colloidal gold (μL) 200 200 200 200 200 200 200 Potassium carbonate (μL) 0 0.4 0.6 0.8 1 2 4 Antibody (μg / mL) 6 6 6 6 6 6 6 10% sodium chloride (μL) 40 40 40 40 40 40 40 Color Change Light purple Pink Purple purple purple purple light pink Light gray

[0056] (2) Labeling: Add equal amount of antibody, vortex mix, and react at 37°C for 1 h;

[0057] (3) Blocking: Add 50 μL of 5% casein solution, vortex to mix, and block at 37°C for 0.5 h;

[0058] (4) Resuspension: Centrifuge at 8000 rpm, 4°C for 10 min, discard the supernatant, add the stock solution, and store in a refrigerator at 4°C;

[0059] (5) Salt precipitation: Add 40 μL of 10% sodium chloride solution to each tube, let it stand for 15 min, take pictures for comparison, and determine the optimal pH value for antibody labeling based on the difference in absorbance values ​​of the UV-visible absorption spectrum.

[0060] The pH value has a great influence on the adsorption of protein by colloidal gold particles because the pH value of the solution affects the net charge of the protein. When the pH is equal to or slightly alkaline to the isoelectric point of the protein, the protein is electrically neutral. At this time, the electrostatic interaction between the protein molecules and the colloidal gold particles is small, the molecular tension is maximum, and it is in a weak hydration state, which makes it easier to adsorb on the surface of the colloidal gold particles. The optimal pH value for colloidal gold labeling was determined by salt precipitation experiments. The pH of the colloidal gold solution was adjusted with 0.1M potassium carbonate solution at a volume ratio of 0:0, 1:500, 3:1000, 1:250, 1:200, 1:100, and 1:50 to the colloidal gold solution, respectively. The amount of labeled antibody was 1 μL, and the antibody diluent was 10 mM phosphate buffer (pH 7.4). The reaction was carried out at 37°C for 1 hour. 10% NaCl was added and allowed to stand for 1 hour to observe the aggregation of the immunogold label precipitation. The results are as follows. Figure 4 As shown, the best effect was achieved by adding 0.1 M potassium carbonate solution at a volume ratio of 3:1000 to the colloidal gold solution.

[0061] (2) Determination of the amount of labeled antibody.

[0062] The dosage of IDH1 mutant monoclonal antibody was 6 μg / mL, the antibody diluent was 10 mM phosphate buffer (pH 7.4), the reaction time was 1 h, the blocking time was 0.5 h, and the pH of the colloidal gold solution was adjusted by adding different amounts of 0.1 M potassium carbonate solution. The specific steps are as follows:

[0063] (1) Adjust pH: Add 200 μL of colloidal gold solution and 0.6 μL of 0.1 M potassium carbonate solution to a centrifuge tube and vortex to mix.

[0064] (2) Labeling: Add antibodies in increasing order from left to right (Table 2), vortex to mix, and react at 37°C for 1 h;

[0065] Table 2

[0066] Test tube number 1 2 3 4 5 6 7 8 Colloidal gold (μL) 200 200 200 200 200 200 200 200 Potassium carbonate (μL) 1 1 1 1 1 1 1 1 Antibody (μg / mL) 0 3 6 9 12 15 18 21 10% sodium chloride (μL) 40 40 40 40 40 40 40 40 Color Change blue-gray blue-gray purple purple purple purple purple purple

[0067] (3) Blocking: Add 50 μL of 5% casein solution, vortex to mix, and block at 37°C for 0.5 h;

[0068] (4) Resuspension: Centrifuge at 8000 rpm, 4°C for 10 min, discard the supernatant, add the stock solution, and store in a refrigerator at 4°C;

[0069] (5) Salt precipitation: Add 40 μL of 10% sodium chloride solution to each tube, let it stand for 15 min, take pictures for comparison, and determine the optimal antibody labeling amount based on the difference in absorbance values ​​of the UV-visible absorption spectrum.

[0070] The amount of antibody used directly impacts the sensitivity and specificity of the test strip. Using too much antibody in a competitive test strip can increase costs and nonspecific binding, leading to false-negative results. Furthermore, excessive amounts of antibody can create excessive background signal on the test strip, affecting the accuracy of the results. Using too little antibody can reduce detection sensitivity and prevent effective capture of the target molecule, resulting in false-positive results.

[0071] The optimal amount of colloidal gold labeled antibody was determined by salt precipitation experiment. The antibody solution was added to the colloidal gold to make the final antibody concentration 0, 3, 6, 9, 12, 15, 18, 21 μg / mL, respectively. The antibody diluent was 10 mM phosphate buffer (pH 7.4), and the reaction was carried out at 37°C for 1 hour. 10% NaCl was added and allowed to stand for 1 hour. The aggregation of the immunogold labeled precipitate was observed. The results are as follows: Figure 5 As shown in the figure, the best effect was achieved when the final concentration was 6 μg / mL.

[0072] (3) Characterization of immunogold label.

[0073] The blocked immunogold-labeled solution was identified and compared with the unblocked immunogold-labeled solution and the unlabeled colloidal gold solution. Figure 2It is a UV-visible spectroscopic spectrum. The colloidal gold solution has an absorption peak at 525nm. When the antibody combines with the colloidal gold, the highest absorption peak shifts to 530nm. After blocking with casein, the highest absorption peak shifts to 531nm. Figure 6 The particle size distribution is as follows: the colloidal gold particle size is 20.7nm, the antibody-colloidal gold conjugate (Au-Ab) particle size is 23.8nm, and the immunogold conjugate after casein blocking (Au-Ab-casein) particle size is 28.1nm. These data demonstrate the successful synthesis of the immunogold conjugate and the successful casein blocking.

[0074] Example 3. Construction of IDH1 mutant colloidal gold immunoassay strips

[0075] The buffer solution and standard solution used in the present invention are as follows:

[0076] The main components of the sample pad treatment solution are: phosphate buffer, Tween-20, polyvinylpyrrolidone-40000, and sucrose. The main reagents are as follows:

[0077]

[0078] Among them, the nucleic acid sequence number of the IDH1 mutant protein is as follows:

[0079] 10 20 30 40 50 60 MSKKISGGSV VEMQGDEMTR IIWELIKEKL IFPYVELDLH SYDLGIENRD ATNDQVTKDA 70 80 90 100 110 120 AEAIKKHNVG VKCATITPDE KRVEEFKLKQ MWKSPNGTIR NILGGTVFRE AIICKNIPRL 130 140 150 160 170 180 VSGWVKPIII GRHAYGDQYR ATDFVVPGPG KVEITYTPSD GTQKVTYLVH NFEEGGGVAM 190 200 210 220 230 240 GMYNQDKSIE DFAHSSFQMA LSKGWPLYLS TKNTILKKYD GRFKDIFQEI YDKQYKSQFE 250 260 270 280 290 300 AQKIWYEHRL IDDMVAQAMK SEGGFIWACK NYDGDVQSDS VAQGYGSLGM MTSVLVCPDG 310 320 330 340 350 360 KTVEAEAAHG TVTRHYRMYQ KGQETSTNPI ASIFAWTRGL AHRAKLDNNK ELAFFANALE 370 380 390 400 410 EVSIETIEAG FMTKDLAACI KGLPNVQRSD YLNTFEFMDK LGENLKIKLA QAKL

[0080] Test strips and other consumables:

[0081] model Consumables Name Manufacturer GL0194 Sample pad Shanghai Jieyi Biotechnology Co., Ltd. H5015 absorbent pads Shanghai Jieyi Biotechnology Co., Ltd. CN140 nitrocellulose membrane Sartorius Scientific Instruments Co., Ltd.

[0082] (1) Assembly of test strips.

[0083] The test strip consists of four basic structural units: sample pad, nitrocellulose membrane, absorbent pad, and base plate, which are overlapped with the base plate according to the direction of sample flow.

[0084] After soaking the sample pad in sample pad treatment solution for 30 minutes, the sample pad was dried in a 37°C oven for 1 hour and cut to a length of 23 mm. The absorbent pad was 18 mm wide. A nitrocellulose membrane was adhered to the center of the baseplate. The sample pad and absorbent pad overlapped the nitrocellulose membrane by 2 mm and adhered to both ends of the baseplate. IDH1 mutant protein (standard) and goat anti-mouse secondary antibody (coating antibody) were diluted to the appropriate concentration in 10 mM phosphate buffer (pH 7.4) and sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm. Two straight lines, T / C, were formed sequentially with a 5 mm spacing. The lines were then dried in a 30°C oven for 1 hour. The test strips were cut into 4 mm strips using a strip cutter. The test strips were then placed in an aluminum foil bag containing a desiccant, sealed, and stored in a refrigerator at 4°C.

[0085] (2) Optimization of test paper conditions.

[0086] (1) Selection of nitrocellulose membrane.

[0087] The thickness, surface properties, pore size, and composition of the nitrocellulose membrane will affect the sensitivity of the test strip. Select NC membranes such as Sartorius CN95, Sartorius CN140, and Pall Vivid170. Spray the prepared T / C line coating solution onto the NC membrane using a film streaker. After drying, assemble it onto the test strip. Compare the experimental results to select the most suitable NC membrane type. Figure 7 The results showed that among the three NC membranes, Sartorius CN140 had the most uniform and moderate color development, so this NC membrane was selected as the material for subsequent experiments.

[0088] (2) Selection of T-line antigen spraying amount.

[0089] The amount of antigen sprayed onto the T line requires precise control. Insufficient spraying may result in unclear color development or a hollow test line. Conversely, excessive spraying may cause the C line to fail to develop color or exhibit dragging, both of which affect the accuracy of the test results. IDH1 mutant protein concentrations of 400, 600, 800, and 1000 μg / mL were sprayed onto the NC membrane using a membrane sprayer at a rate of 1 μL / cm. After drying, the membrane was assembled onto a test strip. The most suitable antigen concentration was selected by comparing the experimental results. Figure 8 The results showed that at a concentration of 1000 μg / mL, the T line of the test strip had a uniform and moderate color development, so this concentration was selected as the parameter for subsequent experiments.

[0090] (3) Selection of the amount of secondary antibody sprayed on line C.

[0091] The amount of secondary antibody sprayed onto the C-line needs to be appropriate to ensure color development during the test. Insufficient application may result in insufficient or no color development on the C-line, making it impossible to confirm the validity of the test. Conversely, excessive application may lead to unnecessary waste of raw materials and increase the production cost of the test strips. Use a membrane streaker to spray a goat anti-mouse secondary antibody at concentrations of 100, 300, 500, and 700 μg / mL onto the NC membrane at a rate of 1 μL / cm. After drying, assemble the membrane onto the test strips. Compare the experimental results to select the most suitable secondary antibody concentration. Figure 9 The results showed that at a concentration of 500 μg / mL, the color of line C on the test strip was uniform and moderate, so this concentration was selected as the parameter for subsequent experiments.

[0092] (4) Selection of the amount of immunogold label.

[0093] Different volumes of immunogold labeled solution were dropped onto the conjugate pad of the test strip. After 1 minute, it was rinsed with running buffer solution containing 1 μg / mL IDH1 mutant protein standard solution. The results were as follows: Figure 10As shown in the figure, when the immunogold conjugate solution exceeds 15 μL, the T-line color change is small, which can easily lead to false-negative results. When the immunogold conjugate solution is less than 15 μL, the T-line color is weak, which is not conducive to quantitative analysis in actual testing. 15 μL of immunogold conjugate solution is the optimal volume, which ensures the intensity of the T-line color while being less likely to cause false-negative results.

[0094] (5) Selection of the time to take a photo of the test strip.

[0095] Select the previously optimized experimental conditions, prepare the test strips and run them, and take a picture of the test strips every 1 minute. Figure 11 As shown, the color of the T / C line increases over time from 1 to 8 minutes, and then the color of the strip stabilizes. When the reaction time is 8 minutes, the T / C line of the test strip is better and the NC membrane background is clear. 8 minutes is the preferred time for imaging after adding the immunogold label.

[0096] (3) Evaluation of test strip detection performance.

[0097] (1) Sensitivity analysis.

[0098] According to the previously optimized experimental conditions, the newly prepared immunogold label solution was added with different concentrations of IDH1 mutant protein standard solution, namely 0μg / mL, 1μg / mL, 5μg / mL, 10μg / mL, 25μg / mL, 50μg / mL, and 100μg / mL. After mixing the above solutions evenly, incubate at room temperature for 10 minutes, drop the mixed solution onto the conjugate pad of the newly prepared test strip, and add the running buffer. The visual results of the test strip are as follows Figure 12 As shown, as the concentration of the IDH1 mutant standard solution increases, the color of the T-line gradually becomes lighter. At a mutant IDH1 protein concentration of 5 μg / mL, the color of the T-line on the test strip undergoes a visible color change. Therefore, the target concentration of 5 μg / mL is the visual detection limit of the test strip.

[0099] The detection results of different concentrations of IDH1 mutant protein standard solution were further processed and analyzed using Image J software. The gray value ratio (relative intensity) of T line and C line was used as the vertical axis, and the different concentrations of IDH1 mutant protein standard solution were used as the horizontal axis. The relationship between the two is as follows: Figure 13 As shown in the figure, within the concentration range of 0-100 μg / mL, the relative intensity value decreases with increasing concentration of the IDH1 mutant protein standard solution, and the test results can be substituted into the linear equation.

[0100] (2) Specificity analysis.

[0101] In order to evaluate the specificity of this method, IDH1 wild-type protein, carcinoembryonic antigen, cardiac troponin, prostate-specific antigen, alpha-fetoprotein, etc. were added to the negative samples for colloidal gold test strip testing at an addition amount of 5 μg / mL. The results are as follows Figure 14 Except for the IDH1 mutant test strip, where the T line became lighter, no significant color change was observed for the other interfering substances, indicating that the test strip has good specificity for the IDH1 mutant protein.

[0102] (3) Actual sample testing.

[0103] First, fresh glioma tissue samples were placed in liquid nitrogen for rapid freezing to ensure that the protein structure and biological activity in the samples were retained to the greatest extent. Subsequently, the frozen tissue samples were ground into a fine powder using a mortar and pestle, and then sonicated for a few seconds in a buffer solution to perform an addition recovery experiment. The results are as follows: Figure 15 As the concentration of IDH1 mutant protein increases, the color of the T line gradually becomes lighter, indicating that the test strip has good sensitivity and responsiveness in actual samples.

[0104] (4) Stability analysis.

[0105] The assembled test strips were placed in an aluminum foil bag with desiccant, vacuumed, and stored at 4°C. They were taken out for testing on the first and thirtieth days. The color intensity and sensitivity of the test strips were as follows: Figure 16 After 30 days of storage under these conditions, the color of the T and C lines of the test strip decreased slightly, but the detection limit did not change much, indicating that the test strip has good stability. The above descriptions are merely a few embodiments of the present invention and are not intended to limit the present invention. Any modifications and equivalent substitutions made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

[0106] The technical means disclosed in the solution of the present invention are not limited to the technical means disclosed in the above-mentioned embodiment, but also include technical solutions composed of any combination of the above technical features.

Claims

1. A colloidal gold immunoassay strip for immediate intraoperative detection of IDH1 mutant protein, comprising a base plate, a sample pad, a nitrocellulose membrane, an absorbent pad, and colloidal gold, characterized in that: The nitrocellulose membrane is provided with a detection T line coated with IDH1 mutant protein and a quality control C line coated with a goat anti-mouse secondary antibody corresponding to the IDH1 mutant monoclonal antibody in sequence along the sample flow direction.

2. A colloidal gold competitive immunochromatographic test strip for detecting IDH1 mutant protein according to claim 1, characterized in that: The distance between the detection T line and the quality control C line is 5-10 mm.

3. A colloidal gold competitive immunochromatographic test strip for detecting IDH1 mutant protein according to claim 1, characterized in that: The nitrocellulose membrane is a Sartorius nitrocellulose membrane CN140 membrane; the colloidal gold test strip is placed in the card cavity in the card housing, and two windows, a sample addition area and a color development area, are provided on the card housing. The sample addition area is aligned with the sample pad area, and the color development area is aligned with the T line and C line areas of the nitrocellulose membrane of the colloidal gold test strip.

4. A method for preparing a colloidal gold competitive immunochromatographic test strip for detecting IDH1 mutant proteins, characterized in that: The specific steps include: Step 1: Preparation of immunogold label: Use 0.1M potassium carbonate solution to adjust the pH of colloidal gold solution, the volume ratio of colloidal gold solution to potassium carbonate solution is 1000:2-10, and mix well; add IDH1 mutant monoclonal antibody, mix well, and react at 25-40°C for 0.5-1.5 hours; add casein solution to a final concentration of 0.5%-1.5%; block for 0.5-1.5 hours, centrifuge, discard the supernatant solution, add reservoir solution, and store in a refrigerator at 4°C; the reservoir solution composition is 5%-15% sucrose and 0.5%-2% casein in phosphate buffer; Step 2: Attach the sample pad, nitrocellulose membrane, and absorbent pad to the base plate in sequence, overlapping each other with an overlap of 2 mm. Step 3: Use a membrane stripper to streak the IDH1 mutant protein and goat anti-mouse secondary antibody onto a nitrocellulose membrane to form a detection T line and a quality control C line; Step 4: Use a strip cutter to cut the base plate and attached materials into test strips with a width of 3-5mm; Step 5: Place the test strip from step 4 into the cartridge. The cartridge has two windows, a sample loading area and a color development area. Align the sample loading area with the sample pad area, and the color development area with the T-line and C-line areas. Step 6: Place the test strips in an aluminum foil bag with desiccant inside and store at 4°C.

5. The method for preparing a colloidal gold competitive immunochromatographic test strip for detecting IDH1 mutant proteins according to claim 4, characterized in that: In step 1, the colloidal gold particles in the colloidal gold solution have a diameter of 20-30 nm.

6. The method for preparing a colloidal gold competitive immunochromatographic test strip for detecting IDH1 mutant proteins according to claim 4, characterized in that: In step (1), the final concentration of the IDH1 mutant monoclonal antibody in the colloidal gold solution is 9 μg / mL.

7. The method for preparing a colloidal gold competitive immunochromatographic test strip for detecting IDH1 mutant protein according to claim 4, characterized in that: In step (2), in the colloidal gold competitive immunochromatographic test strip, the length of the nitrocellulose membrane is 2.5-3 cm, the length of the sample pad is 2-2.5 cm, and the length of the absorbent pad is 1.5-2 cm.

8. The method for preparing a colloidal gold competitive immunochromatographic test strip for detecting IDH1 mutant protein according to claim 4, characterized in that: The protein concentration of the detection T-line coating is 0.6-3 mg / mL, and the protein spraying volume of the detection T-line coating is 0.5-2 μL / cm.

9. The method for preparing a colloidal gold competitive immunochromatographic test strip for detecting IDH1 mutant protein according to claim 4, characterized in that: The protein concentration of the quality control C line coating is 0.3-2 mg / mL, and the protein spraying volume of the quality control C line coating is 0.5-2 μL / cm.

10. Use of the colloidal gold competitive immunochromatographic test strip according to any one of claims 1 to 9 in mutant / wild-type typing detection of brain glioma.

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