A diagnostic reagent for alpha-fetoprotein detection

Abstract

The present invention discloses a diagnostic reagent for alpha-fetoprotein detection, which is prepared into an alpha-fetoprotein latex kit, and positively detected by latex enhanced immunoturbidimetry. In a homogeneous liquid system, antigen and antibody are combined. When the combination ratio is appropriate, a solid immune complex is formed and precipitated from the liquid phase, which is manifested as a change in turbidity. The turbidity change is detected by a fully automatic biochemical analyzer, and a linear curve is drawn using a standard product to obtain the content of the substance to be tested in the sample. The diagnostic reagent has the advantages of convenience, speed, time saving, automatic operation, sensitivity, accuracy, etc., and can meet the needs of clinical large sample detection.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a diagnostic reagent for alpha-fetoprotein detection. Background Art

[0002] Alpha-fetoprotein (α-fetoprotein, αFP or AFP) is a glycoprotein. Under normal circumstances, this protein mainly comes from embryonic liver cells and is present in very low levels in adult serum. Alpha-fetoprotein is closely related to the occurrence and development of liver cancer and various tumors. It can be found in high concentrations in various tumors and can be used as a positive detection indicator for various tumors. At present, it is mainly used as a serum marker for primary liver cancer in clinical practice for the diagnosis and efficacy monitoring of primary liver cancer. As the only hepatocellular carcinoma tumor marker currently recommended for routine clinical use, it is clinically recommended that patients with hepatitis B or C cirrhosis should be followed up for AFP every 6 months. For those with AFP>20ng / ml and a continuous increase, further examination is required even if the abdominal ultrasound is negative. Other malignant tumors such as testicular cancer, teratoma, gastric cancer, pancreatic cancer, etc. can also lead to elevated AFP levels. Therefore, there is a great demand for highly sensitive and specific AFP diagnostic kits in clinical practice.

[0003] Traditional LETIA uses polyclonal antibodies to coat latex particles for detection. However, polyclonal antibodies can recognize multiple epitopes of antigens and produce non-specific binding antibodies, resulting in a high reaction background and affecting the specificity of the detection. Due to individual differences in animals, polyclonal antibodies are prone to batch differences. Utility Model Content

[0004] The purpose of the present invention is to solve the problems of high cost, slow detection speed and low accuracy, and to propose a diagnostic reagent for alpha-fetoprotein detection.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme: the preparation of the diagnostic reagent for alpha-fetoprotein detection comprises the following steps:

[0006] S1: Preparation of mouse monoclonal antibody against alpha-fetoprotein;

[0007] (1) Animal immunization: Purchase 95% pure human AFP protein, mix it with nano-adjuvant, and immunize 3 8-week-old Balb / C mice. Immunization is performed by multi-point injection on the back, with 100ug antigen / mouse as the main injection and 50ug antigen / mouse as the booster injection. After 5 immunizations, the titer is tested by indirect ELISA, and the mouse with the highest titer is selected for fusion;

[0008] (2) Myeloma cell preparation: One week before fusion, resuscitate SP2 / 0 cells and culture normally to the logarithmic phase;

[0009] (3) Preparation of spleen cells: Select the mice to be fused, kill them by cervical dislocation on the day of fusion, remove the spleen, and collect and count spleen cells according to standard procedures;

[0010] (4) Cell fusion: 1) Mix myeloma cells and spleen cells at a ratio of 1:3-1:10, perform cell fusion operation according to standard procedures, and then culture with HAT DMEM complete medium. Hybridoma cells can be seen 3 days after fusion. Change 1 / 2HAT complete medium on the 7th day and 1 / 2HT medium on the 8th day. Screening and detection began about 10 days after fusion. After fusion, culture with HAT selective medium. Observe under a microscope and see multiple growing hybridoma cells, proving that the fusion operation was successful. 2) Take 100ul / well of cell supernatant for indirect ELISA detection. According to the ELISA results, judge the positive wells, use a single-channel pipette to pick the positive wells detected on the whole plate, and conduct a second re-examination to further confirm the positive wells; 3) Perform two rounds of subcloning on the cells in the re-screened positive wells. The cells in the positive wells were subcloned by limiting dilution for the first time, and then transferred to multiple wells. The cells were cultured with HT DMEM medium. The cells were observed under a microscope for about 7 days. The wells with clone growth were detected by indirect ELISA. The wells with high OD values ​​were taken as positive wells. The cells in the positive wells were picked for the second subcloning. Stable positive hybridoma cell lines were detected as the cells for the final preparation of monoclonal antibodies, and the culture was expanded. 4) The above positive cells were expanded and cultured and injected into the abdominal cavity of Balb / C mice (sensitized with Freund's incomplete adjuvant). Generally, the abdominal bulge of the mice was seen in 7-10 days, indicating that ascites was produced. When the mice had obvious ascites, the ascites was extracted in time. 5) The ascites of the above cells was purified with ProteinA / G column. The purity of the purified antibodies was greater than 90%. After the concentration purity was tested, the concentration was adjusted to facilitate storage.

[0011] S2: AFP monoclonal antibodies were paired in pairs;

[0012] (1) Biotin labeling operation process: 1) The purified AFP monoclonal antibody was dialyzed three times with carbonate buffer (0.1ML, NaHCO3 / Na2CO3, pH9.5, containing 0.1% NaN3) at 2-8°C. After the dialysis, the immunoglobulin concentration was adjusted to 20 mg / mL; 2) NHS-D-Biotin was dissolved with DMSO (dimethyl sulfoxide), and the concentration of NHS-D-Biotin was adjusted to 22 mg / mL. The mixture was divided into small tubes and stored at -20°; 3) The antibody to be labeled and NHS-D-Biotin were mixed at a volume ratio of 10:1. The mixture was kept at room temperature, away from light, stirred gently, and reacted for 4 hours; 4) The reaction solution was dialyzed three times with PBS buffer (0.01M phosphate solution, 0.15M sodium chloride solution, pH7.4, containing 0.1% NaN3) at 2-8°C; 5) After the dialysis, the biotinylated antibody was stored at -20°C.

[0013] (2) Monoclonal antibody pairing: 1) Coat the strips with unlabeled antibody (Cap-ab) at a dilution of 1:1000, 100ul / well, and incubate at 4°C overnight; 2) Shake out the coating solution, wash 2-3 times with washing solution (300uL / well), pour out the washing solution, tap 10 times to ensure that there is no residual liquid in the well; 3) Add blocking solution (300uL / well) to seal the strips, seal at 37°C for 2 hours, shake out the blocking solution, and wash twice with washing solution; 4) Add antigen, 50ng / well for positive test plate and 50ng / well for blank control plate No antigen was added, but the diluent was used instead (0 ng / well), and the plates were incubated at 37° for 1 hour; the liquid in the plate was shaken off, and the plates were washed 4 times with washing solution; 5) biotin-labeled detection antibody (Det-ab) was added, with a dilution of 1:1000, at 100 uL / well, and the plates were incubated at 37°C for 1 hour; the liquid in the plate was shaken off, and the plates were washed 4 times with washing solution; 6) secondary antibody was added, and HRP-labeled streptavidin was diluted 1:10000, and added to the wells, at 100 uL / well, and the plates were incubated at 37°C for 1 hour; the liquid in the plate was shaken off, and the plates were washed 4 times with washing solution.

[0014] (3) Draw conclusions on monoclonal antibody pairing.

[0015] S3: Preparation of AFP Latex Enhancement Kit

[0016] (1) Preparation process of latex R2: 1) Take carboxyl latex microspheres with a particle size of 270nm, wash 1ml of microspheres (100mg / ml) twice with activation reagent MES (pH6.0.50mmol), and wash with 5ml solution each time; after the second activation, resuspend the microspheres in 4.5ml of activation reagent to ensure that the microspheres are evenly dispersed; 2) Add 2mg EDAC (final concentration 0.4mg / ml, dissolved in pH6.0, 0.50mmol MES) and 9mg NHS (final concentration 1.8mg / m, dissolved in pH6.0, 0.05M MES solution) while mixing; 3) Mix and react for 60min (18-37 degrees); 4) Wash twice with H6.0, 0.05M MES solution (centrifugal speed 15000 rpm, 20min), and finally suspend in 5ml pH6.0, 0.50mmol MES, to ensure that the microspheres are evenly dispersed; 5) Add appropriate amount of antibody, mix the microspheres and protein, and react at room temperature for 2 to 4 hours; 6) Add blocking reagent Gly to a final concentration of 40mM, ethanolamine to a final concentration of 40mM, and react for 60 minutes; Add BSA to a final concentration of 1% and react overnight; 7) pH7.4 PBS + 0.1% Tween solution, wash the microspheres three times, and ultrasonically disperse the microspheres each time; 8) Suspend the microspheres in PBS + 0.1% Tween solution, 0.01% preservative, 1% BSA, and store at 4°C;

[0017] (2) Prepare an alpha-fetoprotein latex test kit.

[0018] In summary, due to the adoption of the above technical scheme, the beneficial effects of the present invention are: by making an alpha-fetoprotein latex kit, positive detection is performed by latex enhanced immunoturbidimetry, and antigen-antibody binding occurs in a homogeneous liquid system. When the binding ratio is appropriate, a solid immune complex is formed and precipitated from the liquid phase, which is manifested as a change in turbidity. This turbidity change is detected by a fully automatic biochemical analyzer, and a linear curve is drawn using a standard product to obtain the content of the substance to be tested in the sample. It has the advantages of convenience, speed, time saving, automated operation, sensitivity, accuracy, etc., and can meet the needs of clinical large sample detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of the linear range of other reagent detection is shown;

[0020] Figure 2 A schematic diagram of the linear range of detection of this reagent is shown. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments 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.

[0022] Example 1: Preparation of mouse monoclonal antibody against alpha-fetoprotein;

[0023] (1) Animal immunization: Purchase 95% pure human AFP protein, mix it with nano-adjuvant, and immunize 3 8-week-old Balb / C mice. Immunization is performed by multi-point injection on the back, with 100ug antigen / mouse as the main injection and 50ug antigen / mouse as the booster injection. After 5 immunizations, the titer is tested by indirect ELISA, and the mouse with the highest titer is selected for fusion;

[0024] (2) Myeloma cell preparation: One week before fusion, resuscitate SP2 / 0 cells and culture normally to the logarithmic phase;

[0025] (3) Preparation of spleen cells: Select the mice to be fused, kill them by cervical dislocation on the day of fusion, remove the spleen, and collect and count spleen cells according to standard procedures;

[0026] (4) Cell fusion: 1) Mix myeloma cells and spleen cells at a ratio of 1:3-1:10, perform cell fusion operation according to standard procedures, and then culture with HAT DMEM complete medium. Hybridoma cells can be seen 3 days after fusion. Change 1 / 2HAT complete medium on the 7th day and 1 / 2HT medium on the 8th day. Screening and detection began about 10 days after fusion. After fusion, culture with HAT selective medium. Observe under a microscope and see multiple growing hybridoma cells, proving that the fusion operation was successful. 2) Take 100ul / well of cell supernatant for indirect ELISA detection. According to the ELISA results, judge the positive wells, use a single-channel pipette to pick the positive wells detected on the whole plate, and conduct a second re-examination to further confirm the positive wells; 3) Perform two rounds of subcloning on the cells in the re-screened positive wells. The cells in the positive wells were subcloned by limiting dilution for the first time, and then transferred to multiple wells. The cells were cultured with HT DMEM medium. The cells were observed under a microscope for about 7 days. The wells with clone growth were detected by indirect ELISA. The wells with high OD values ​​were taken as positive wells. The cells in the positive wells were picked for the second subcloning. Stable positive hybridoma cell lines were detected as the cells for the final preparation of monoclonal antibodies, and the culture was expanded. 4) The above positive cells were expanded and cultured and injected into the abdominal cavity of Balb / C mice (sensitized with Freund's incomplete adjuvant). Generally, the abdominal bulge of the mice was seen in 7-10 days, indicating that ascites was produced. When the mice had obvious ascites, the ascites was extracted in time. 5) The ascites of the above cells was purified with ProteinA / G column. The purity of the purified antibodies was greater than 90%. After the concentration purity was tested, the concentration was adjusted to facilitate storage.

[0027] Example 2: AFP monoclonal antibodies are paired in pairs;

[0028] (1) Biotin labeling operation process: 1) The purified AFP monoclonal antibody was dialyzed three times with carbonate buffer (0.1ML, NaHCO3 / Na2CO3, pH9.5, containing 0.1% NaN3) at 2-8°C. After the dialysis, the immunoglobulin concentration was adjusted to 20 mg / mL; 2) NHS-D-Biotin was dissolved with DMSO (dimethyl sulfoxide), and the concentration of NHS-D-Biotin was adjusted to 22 mg / mL. The mixture was divided into small tubes and stored at -20°; 3) The antibody to be labeled and NHS-D-Biotin were mixed at a volume ratio of 10:1. The mixture was kept at room temperature, away from light, stirred gently, and reacted for 4 hours; 4) The reaction solution was dialyzed three times with PBS buffer (0.01M phosphate solution, 0.15M sodium chloride solution, pH7.4, containing 0.1% NaN3) at 2-8°C; 5) After the dialysis, the biotinylated antibody was stored at -20°C.

[0029] (2) Monoclonal antibody pairing: 1) Coat the strips with unlabeled antibody (Cap-ab) at a dilution of 1:1000, 100ul / well, and incubate at 4°C overnight; 2) Shake out the coating solution, wash 2-3 times with washing solution (300uL / well), pour out the washing solution, tap 10 times to ensure that there is no residual liquid in the well; 3) Add blocking solution (300uL / well) to seal the strips, seal at 37°C for 2 hours, shake out the blocking solution, and wash twice with washing solution; 4) Add antigen, 50ng / well for positive test plate and 50ng / well for blank control plate No antigen was added, but the diluent was used instead (0 ng / well), and the plate was incubated at 37° for 1 hour; the liquid in the plate was shaken off, and the plate was washed 4 times with washing solution; 5) Biotinylated detection antibody (Det-ab) was added, with a dilution of 1:1000, 100uL / well, and the plate was incubated at 37°C for 1 hour; the liquid in the plate was shaken off, and the plate was washed 4 times with washing solution; 6) Secondary antibody was added, HRP-labeled streptavidin was diluted 1:10000, and added to the wells, 100uL / well, and the plate was incubated at 37°C for 1 hour; the liquid in the plate was shaken off, and the plate was washed 4 times with washing solution;

[0030] (3) Draw the conclusion of monoclonal antibody pairing: 1) Color development: Add TMB substrate to all wells, 100uL / well, and the substrate is color developed. Place at 37° for 15-30 minutes, and the color turns blue; 2) Add 2M H2SO4 (50uL / well) stop solution to terminate the reaction. The color turns yellow after termination; 3) Place the ELISA plate on the ELISA reader, select the corresponding program, and read the OD450 nm value. The pairing results are shown in the table below:

[0031]

[0032] Select monoclonal antibodies of hybridoma strains 49, 51, and 62 as raw materials to prepare AFP latex reagent

[0033] Example 3: Preparation of Alpha-fetoprotein Latex Enhanced Kit

[0034] (1) Preparation process of latex R2: 1) Take carboxyl latex microspheres with a particle size of 270 nm, wash 1 ml of microspheres (100 mg / ml) twice with activation reagent MES (pH 6.0.50 mmol), and wash with 5 ml of solution each time; after the second activation, resuspend the microspheres in 4.5 ml of activation reagent to ensure that the microspheres are evenly dispersed; 2) Add 2 mg EDAC (final concentration 0.4 mg / ml, dissolved in pH 6.0, 0.50 mmol MES) and 9 mg NHS (final concentration 1.8 mg / m, dissolved in pH 6.0, 0.05 M MES solution) while mixing;

[0035]

[0036]

[0037] 3) Mix and react for 60 minutes (18-37 degrees); 4) Wash twice with H6.0, 0.05M MES solution (centrifugal speed 15000 rpm, 20 minutes), and finally suspend in 5ml pH6.0, 0.50mmol MES to ensure that the microspheres are evenly dispersed; 5) Add appropriate amount of antibody, mix the microspheres and protein, and react at room temperature for 2-4 hours; 6) Add blocking reagent Gly final concentration 40mM, ethanolamine final concentration 40mM, mix and react for 60 minutes; add BSA, final concentration 1%, and react overnight; 7) pH7.4 PBS + 0.1% Tween solution, wash the microspheres three times, and ultrasonically disperse the microspheres each time; 8) Suspend the microspheres in PBS + 0.1% Tween solution, 0.01% preservative, 1% BSA, and store at 4°C;

[0038] (2) Performance test of the prepared alpha-fetoprotein latex test kit.

[0039]

[0040]

[0041] Batch-to-batch precision 1 2 Mean (mg / L) 20.8 198.8 SD 0.324 0.552 CV(%) 1.56 0.27 n 20 20

[0042] Intra-batch precision 1 2 Mean (mg / L) 19.9 201.2 SD 0.231 0.496 CV(%) 1.16 0.25 n 20 20

[0043] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and utility model concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A diagnostic reagent for detecting alpha-fetoprotein, characterized in that: The preparation of the diagnostic reagent for alpha-fetoprotein detection includes the following steps: S1: Preparation of mouse monoclonal antibody against alpha-fetoprotein; (1) Animal immunization: Human AFP protein with a purity of 95% was purchased and mixed with nano-adjuvants. Three 8-week-old Balb / C mice were immunized by multiple injections on the back. The main injection was 100 ug antigen per experimental mouse, and the booster injection was 50 ug antigen per experimental mouse. After 5 immunizations, the titer was tested by indirect ELISA, and the mouse with the highest titer was selected for fusion; (2) Myeloma cell preparation: One week before fusion, resuscitate SP2 / 01 cells and culture normally to the logarithmic phase; (3) Preparation of spleen cells: Select the mice to be fused, kill them by cervical dislocation on the day of fusion, remove the spleen, and collect and count spleen cells according to standard procedures; (4) Cell fusion: 1) Myeloma cells and spleen cells were mixed at a ratio of 1:3-1:10, and the cell fusion operation was performed according to the standard process. Then, HAT DMEM complete medium was used for culture. Hybridoma cells could be seen 3 days after fusion. 1 / 2HAT complete medium was replaced on the 7th day, and 1 / 2HT medium was replaced on the 8th day. Screening and detection began about 10 days after fusion. After fusion, HAT selective medium was used for culture. Multiple growing hybridoma cells were observed under a microscope, proving that the fusion operation was successful; 2) 100 μl / well of cell supernatant was aspirated for indirect ELISA detection; based on the ELISA results, the positive wells were judged, and the positive wells detected on the whole plate were picked with a single-channel pipette for a second re-inspection to further confirm the positive wells; 3) Two rounds of subcloning were performed on the re-screened positive wells; in the first subcloning, the cells in the positive wells were limitedly diluted to multiple wells, and HT was added. Cultivate in DMEM medium, observe under a microscope for about 7 days, detect the wells with clone growth by indirect ELISA, and take the wells with high OD values ​​as positive wells; pick the cells in the positive wells for a second subcloning, detect the stable positive hybridoma cell lines, use them as the cells for the final preparation of monoclonal antibodies, and expand the culture; 4) expand the culture of the above stable positive hybridoma cell lines and inject them into the peritoneal cavity of Balb / C mice, that is, the peritoneal cavity of mice sensitized with Freund's incomplete adjuvant. Generally, the bulge of the mouse abdomen can be seen in 7-10 days, which means that ascites is produced; when the mouse has obvious ascites, extract the ascites in time; 5) purify the ascites of the above cells with ProteinA / G column, and the purity of the purified antibody is greater than 90%; after detecting the concentration purity, adjust the concentration to facilitate storage; S2: AFP monoclonal antibodies were paired in pairs; (1) Biotin labeling operation process: 1) The purified AFP monoclonal antibody is dialyzed three times at 2-8°C with carbonate buffer, wherein the carbonate buffer is 0.1mL, contains NaHCO3 and Na2CO3, has a pH of 9.5, and contains 0.1% NaN3. After the dialysis, the immunoglobulin concentration is adjusted to 20 mg / mL; 2) NHS-D-Biotin is dissolved in DMSO dimethyl sulfoxide solution, and the operation is protected from light. The concentration of NHS-D-Biotin is adjusted to 2 2mg / mL, divided into small tubes, stored at -20°; 3) Mix the antibody to be labeled with NHS-D-Biotin in a volume ratio of 10:1; react at room temperature, away from light, gently stir, and react for 4 hours; 4) The solution after the reaction is dialyzed three times at 2-8°C with PBS buffer, wherein the PBS buffer contains 0.01mL phosphate solution and 0.15mL sodium chloride solution, pH 7.4, and contains 0.1% NaN3; 5) After the dialysis, store the biotinylated antibody at -20°C; (2) Monoclonal antibody pairing: 1) Coat the plate with unlabeled antibody Cap-ab, dilution 1:1000, 100 μl / well, overnight at 4°C; 2) Shake out the coating solution, wash 2-3 times with washing solution, 300 μl / well, pour out the washing solution, tap 10 times to ensure that there is no residual liquid in the well; 3) Add blocking solution, 300 μl / well to block the plate, seal at 37°C for 2 hours, shake out the blocking solution, wash twice with washing solution; 4) Add antigen, 50 ng / well for positive test plate, and 50 ng / well for blank control plate No antigen was added, but the diluent was used instead, ng / well, and incubated at 37°C for 1h; the liquid in the plate was shaken off and washed 4 times with washing solution; 5) Biotinylated detection antibody Det-ab was added, dilution 1:1000, 100μl / well, and incubated at 37°C for 1h; the liquid in the plate was shaken off and washed 4 times with washing solution; 6) Secondary antibody was added, HRP-labeled streptavidin was diluted 1:10000, added to the well, 100μl / well, and incubated at 37°C for 1h; the liquid in the plate was shaken off and washed 4 times with washing solution; (3) Draw conclusions on monoclonal antibody pairing; S3: Preparation of AFP Latex Enhancement Kit (1) Preparation process of latex R2: 1) Take carboxyl latex microspheres with a particle size of 270nm, wash 1ml of microspheres twice with activation reagent MES, pH6.0, 50mmol, 100mg / ml, and wash with 5ml solution each time; after the second activation, resuspend the microspheres in 4.5ml of activation reagent to ensure that the microspheres are evenly dispersed; 2) Add 2mg EDAC, final concentration of 0.4mg / ml, dissolved in pH6.0, 0.50mmol MES and 9mg NHS, final concentration of 1.8mg / m, dissolved in pH6.0, 0.05M MES solution while mixing; 3) Mix and react for 60min at 18-37 degrees; 4) Wash twice with H6.0, 0.05M MES solution, centrifuge at 15000 rpm for 20min, and finally suspend in 5ml pH6.0, 0.50mmol MES, to ensure that the microspheres are evenly dispersed; 5) Add appropriate amount of antibody, mix the microspheres and protein, and react at room temperature for 2 to 4 hours; 6) Add blocking reagent Gly, final concentration 40mM, ethanolamine final concentration 40mM, and mix for 60 minutes; Add BSA, final concentration 1%, and react overnight; 7) pH7.4 PBS + 0.1% Tween solution, wash the microspheres three times, and ultrasonically disperse the microspheres each time; 8) Suspend the microspheres in PBS + 0.1% Tween solution, 0.01% preservative, 1% BSA, and store at 4°C; (2) Prepare an alpha-fetoprotein latex test kit.

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