Sample treating fluid, vitamin B12 detection sample pretreatment method, detection kit, detection method and application of vitamin B12 detection sample pretreatment method

Abstract

The invention discloses a sample treating fluid, a vitamin B12 detection sample pretreatment method, a detection kit, a detection method and application thereof, and particularly discloses the sample treating fluid which comprises strong base and sodium dodecyl sarcosinate. According to the sample treatment liquid, VB12 can be completely dissociated, and a relatively stable state can be kept. The invention further provides a detection sample pretreatment method of the vitamin B12. The detection sample pretreatment method comprises the step of mixing the sample treatment liquid with a to-be-detected sample for reaction. According to the sample treatment liquid provided by the invention, VB12 can still be dissociated from binding protein under the condition that highly toxic cyanide or a complex containing cyanide ions and unstable in dissociation is not adopted, and the VB12 can be kept in a relatively stable state to participate in subsequent reaction.

Description

Technical Field

[0001] This invention belongs to the field of immunodiagnostic technology, specifically relating to a sample processing solution and vitamin B. 12 The sample pretreatment methods, test kits, test methods and their applications. Background Technology

[0002] Vitamin B 12 (VB) 12 Vitamin B1 is a water-soluble vitamin, also known as cobalamin because it contains cobalt. The presence of cobalt makes vitamin B1... 12 The crystals are red, therefore VB 12 Also known as the red vitamin. VB 12 It is the most recently discovered B vitamin to date, and the only one containing a metal element. Its main structure is a corrin ring with a Co atom at the center. (VB) 12 Its molecular formula is CoC 63 H 88 N 14 Vitamin P, with a relative molecular weight of 1355.38, is the vitamin with the largest molecular weight and the most complex structure among the B vitamins. (VB) 12 It is readily soluble in water and ethanol, but unstable in strong acid or strong alkali solutions. (Vitamin B) 12 The main form of vitamin B in plasma is methylcobalamin (MeCbl), which is usually bound to transcobalamin proteins. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 12 Approximately 90% is stored as transcobalamin I, with the remaining 10% transported physiologically by transcobalamin II. This binding form helps with vitamin B12. 12 Stable presence and transport in blood plasma.

[0003] Currently testing for vitamin B 12 Immunological methods mainly include radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), and chemiluminescent immunoassay. All of these detection methods require sample pretreatment to remove VB. 12 The VB needs to be dissociated from the binding protein and converted into a stable cyanocobalamin form for subsequent concentration measurement. Imported commercially available test kits typically use a dissociation solution in their sample pretreatment consisting mainly of a strong base, the reducing agent dithiothreitol (DTT), and cyanide. The strong base and DTT disrupt the binding protein structure, allowing the VB to dissociate. 12Sodium cyanide and potassium cyanide are released and form stable cyanocobalamin with cyanide. However, both sodium cyanide and potassium cyanide are highly toxic compounds, subject to strict regulation in their use, making experimental operations difficult and posing a potential environmental pollution risk. Existing methods use cyanide-containing ion complexes to replace cyanide. These complexes are difficult to dissociate into highly toxic cyanide ions, thus exhibiting low toxicity and not being classified as controlled compounds. However, cyanide-containing ion complexes also suffer from low and unstable dissociation degrees, which can adversely affect detection results. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a sample processing solution that enables VB... 12 It completely dissociates and can maintain a relatively stable state.

[0005] This invention also proposes a vitamin B 12 The sample pretreatment method for detection.

[0006] This invention also proposes a vitamin B 12 Test kit.

[0007] This invention also proposes a vitamin B 12 The detection method.

[0008] The present invention also proposes the application of the above-mentioned sample processing solution.

[0009] According to a first aspect of the present invention, a sample processing solution is provided, characterized in that the sample processing solution comprises a strong base and sodium dodecyl sarcosinate.

[0010] In some embodiments of the present invention, the sample processing solution comprises 5-100 mg / mL of a strong base and 1-20 mg / mL of sodium dodecyl sarcosinate.

[0011] In some embodiments of the present invention, the sample processing solution comprises 5-50 mg / mL of a strong base and 1-5 mg / mL of sodium dodecyl sarcosinate.

[0012] In some embodiments of the present invention, the strong alkali includes any one of lithium hydroxide, sodium hydroxide, and potassium hydroxide.

[0013] According to a second aspect of the present invention, a vitamin B12 is provided. 12 The sample pretreatment method for detection includes: mixing and reacting the sample processing solution described in the first aspect of the present invention with the sample to be tested.

[0014] In some embodiments of the present invention, the mixing volume ratio of the sample processing solution to the sample to be tested is 1:(1~8).

[0015] In some embodiments of the present invention, the mixing volume ratio of the sample processing solution to the sample to be tested is 1:(1~3).

[0016] In some embodiments of the present invention, the reaction time is 5 to 30 minutes.

[0017] In some embodiments of the present invention, the reaction time is 5 to 15 minutes.

[0018] In some embodiments of the present invention, the reaction temperature is 34°C to 40°C.

[0019] According to a third aspect of the present invention, a vitamin B12 is provided. 12 The test kit, containing vitamin B 12 The test kit includes: the sample processing solution described in the first aspect of the present invention, streptavidin-labeled magnetic beads, intrinsic factor-labeled biotin, labeled enzyme, and chemiluminescent substrate solution.

[0020] In some embodiments of the present invention, the labeling enzyme includes vitamin B. 12 Derivative-labeled alkaline phosphatase.

[0021] In some embodiments of the present invention, the vitamin B 12 The molar ratio of the derivative to alkaline phosphatase is 1:(1~5).

[0022] In some embodiments of the present invention, the chemiluminescent substrate liquid includes AMPPD or APS-5.

[0023] In some embodiments of the present invention, the mass ratio of streptavidin to magnetic beads in the streptavidin-labeled magnetic beads is 1:(40~60).

[0024] In some embodiments of the present invention, the molar ratio of intrinsic factor to biotin in the intrinsic factor-labeled biotin is 1:(10~80).

[0025] In some embodiments of the present invention, the molar ratio of intrinsic factor to biotin in the intrinsic factor-labeled biotin is 1:(40~60).

[0026] In some embodiments of the present invention, the vitamin B 12 The test kit also includes a cleaning solution.

[0027] In some embodiments of the present invention, the cleaning solution comprises: 8-12 mmol / L HEPES buffer (pH 7.0), with the addition of 0.05%-0.2% (v / v) Tween 20 and 0.3-0.8 g / L sodium azide.

[0028] According to a fourth aspect of the present invention, a vitamin B12 is provided. 12 The detection method, wherein the detection method adopts the vitamin B3 method described in the third aspect of the present invention. 12 The test kit performs the test, which includes the following steps: S1: Mix the sample to be tested with the sample processing solution at a volume ratio of (1~8):1 and react thoroughly. S2: Add biotin labeled with intrinsic factor to the reaction system in step S1 and mix well for reaction; S3: Add streptavidin-labeled magnetic beads and labeled enzyme to the reaction system in step S2 and mix well. S4: Perform magnetic separation, wash with cleaning solution 2-4 times, add chemiluminescent substrate solution, and then perform photoelectric signal interpretation.

[0029] In some embodiments of the present invention, the intrinsic factor-labeled biotin in step S2 is diluted to a working solution of 0.01-0.2 μg / mL before addition; the diluent used includes 200-300 mmol / L MES buffer (pH 5.0).

[0030] In some embodiments of the present invention, the volume ratio of the working solution of the intrinsic factor-labeled biotin to the sample to be tested is 1:1.

[0031] In some embodiments of the present invention, the streptavidin-labeled magnetic beads described in step S3 are diluted to a working solution of 0.1 to 0.5 mg / mL before addition; the diluent used includes a 40 to 60 mmol / L MOPS buffer (pH 7.4) containing 1 to 3 g / dL BSA, 0.5 to 2 g / dL sucrose and 0.01 to 0.03 g / dL sodium azide.

[0032] In some embodiments of the present invention, the volume ratio of the working solution of the streptavidin-labeled magnetic beads to the sample to be tested is 1:1.

[0033] In some embodiments of the present invention, the labeling enzyme described in step S3 is diluted to a working solution of 0.01~0.2 μg / mL before addition, and the diluent used includes 40~60 mmol / L MES (pH 5.0) buffer.

[0034] In some embodiments of the present invention, the cleaning solution comprises: 8-12 mmol / L HEPES buffer (pH 7.0), with the addition of 0.05%-0.2% (v / v) Tween 20 and 0.3-0.8 g / L sodium azide.

[0035] In some embodiments of the present invention, the reaction time in step S1 is 5 to 30 minutes and the reaction temperature is 34°C to 40°C.

[0036] In some embodiments of the present invention, the reaction time in steps S2 and S3 is 3 to 8 minutes and the reaction temperature is 34°C to 40°C.

[0037] The present invention has at least the following beneficial effects: The sample processing solution provided by this invention combines a strong base reagent with sodium dodecyl sarcosinate, which can make VB... 12 It completely dissociates and remains in a relatively stable state. Sodium dodecyl sarcosinate is an amino acid-based anionic surfactant. Its amphoteric properties allow it to neutralize some alkalinity. Simultaneously, sodium dodecyl sarcosinate can form micelle structures that can encapsulate the dissociated vitamin B1. 12 Molecules, thereby reducing VB 12 Direct contact with water molecules reduces the risk of hydrolysis. The sample processing solution provided by this invention, without using highly toxic cyanide or unstable cyanide-containing complexes, can still allow VB to form. 12 It dissociates from the binding protein and remains in a relatively stable state to participate in subsequent reactions.

[0038] Vitamin B provided by this invention 12 The test kit and corresponding test method can not only enable VB 12 It rapidly dissociates from binding proteins and maintains a relatively stable state for subsequent detection reactions. The concentrations of the test samples measured using this kit and detection method show a high correlation with those measured using commercially available reagents. In a weakly acidic environment (pH 4.5–5.0), vitamin B... 12 The optimal stability is achieved by adjusting the pH of the buffer solution to the range of 4.5–5.0 in the subsequent reaction step, which will maintain the VB. 12 The stability of the molecule provides suitable reaction conditions for subsequent reactions. The detection method provided by this invention has good linearity, high accuracy, and high repeatability, and is simple and rapid to operate, making it widely applicable to the detection of VB in clinical samples. 12 The detection method is environmentally friendly. Attached Figure Description

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a correlation result graph from the experimental examples of the present invention. Detailed Implementation

[0040] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0041] Unless otherwise specified, all reagents and instruments used in the following examples are commercially available reagents and instruments obtained through conventional purchasing channels. Example

[0042] This embodiment provides a method for detecting vitamin B. 12 The sample processing solution comprises 2% sodium hydroxide as a releasing agent and 0.2% sodium dodecyl sarcosinate as a surfactant, and its preparation method is as follows: Weigh 20 g of sodium hydroxide, add it to 800 mL of purified water and stir to dissolve. Then add 2 g of sodium dodecyl creatine and stir to dissolve. Finally, bring the volume of the system to 1000 mL with purified water to obtain the sample processing solution. Example

[0043] This embodiment provides a vitamin B 12 The sample pretreatment method for detection uses the sample processing solution provided in Example 1 to pretreat the sample to be tested. The specific steps are as follows: Mix the sample to be tested with the sample processing solution at a volume ratio of 2:1 and react at 37°C for 10 min. Example

[0044] This embodiment provides a vitamin B 12 The test kit includes: the sample processing solution provided in Example 1, streptavidin-labeled magnetic beads, biotin labeled with intrinsic factor, alkaline phosphatase, washing solution, and substrate solution.

[0045] The preparation methods for streptavidin-labeled magnetic beads, intrinsic factor-labeled biotin, alkaline phosphatase, washing solution, and substrate solution are as follows: 1) Streptavidin protein-labeled magnetic beads: Vortex the carboxyl magnetic bead solution (bead size 2.8 μm) and transfer 1 mL of the solution (containing 10 mg of carboxyl magnetic beads) to a centrifuge tube; magnetically separate for 1 minute and carefully remove the supernatant. Add 1 mL of 10 mmol / L LMES buffer (pH 5.5) to the carboxyl magnetic beads and mix thoroughly. Then add 100 μL of EDC solution (20 mg / mL) and vortex at room temperature for 30 minutes. Add 200 μg of streptavidin (SA) and vortex at room temperature for 2 hours. Then magnetically separate and remove the supernatant. Add 1 mL of 1% BSA solution and vortex at room temperature for 30 minutes. Then magnetically separate and remove the supernatant. Add 1 mL of washing buffer, vortex, and magnetically separate to remove the supernatant. Repeat the washing process three times. Finally, add the preservation solution to the modified magnetic beads and store at 2℃–8℃. Before use, dilute the streptavidin-labeled magnetic beads to a working concentration of 0.3 mg / mL with diluent. The amount of protein labeled in the streptavidin-labeled magnetic beads is 20 μg SA protein corresponding to 1 mg of magnetic beads.

[0046] The washing buffer was a 20 mmol / L Tris buffer (pH 7.4) containing 0.1% (v / v) Tween 20, the storage buffer was a 20 mmol / L Tris buffer (pH 7.4) containing 0.2 g / dL BSA, 0.1% (v / v) Tween 20 and 0.2% (v / v) Proclin 300, and the dilution buffer was a 50 mmol / L MOPS buffer (pH 7.4) containing 2 g / dL BSA, 1 g / dL sucrose and 0.02 g / dL sodium azide.

[0047] 2) Biotin labeled with intrinsic factor: 0.1 mg of intrinsic factor protein and biotin-N-hydroxysulfosuccinimide (NHS) were mixed at a molar ratio of 1:50 and reacted at room temperature for 1 hour. After the reaction, unbound biotin-N-hydroxysulfosuccinimide was removed by dialyzing. The intrinsic factor-labeled biotin was then aspirated and added to a preservation solution (based on the preservation solution in step 1 with 50% glycerol added) and stored at -20°C. Before use, the intrinsic factor-labeled biotin (0.1 mg / mL) was diluted 1000 times with 250 mmol / L MES buffer (pH 5.0) to prepare an intrinsic factor-labeled biotin working solution (0.1 μg / mL).

[0048] 3) VB 12 Derivative alkaline phosphatase labeling: Dissolve 7.15 mg of 4-maleimide butyrate-N-hydroxysuccinimide ester (GMBS) in 3.6 mL of DMSO; dissolve alkaline phosphatase (AP) in PBS and adjust the concentration to 1 mg / mL; mix the GMBS and AP solutions and incubate at 37°C for 30 minutes; then transfer the solution to a dialysis bag and dialyze against PBS at pH 7.0 for 4 hours. Collect the dialysate and concentrate it to 10 mg / mL for later use. Add 1 mg of vitamin B... 12 - Adjust the BSA concentration to 2 mg / mL with PBS solution, and add 50 μL of 10 mmol / L N-succinimide-S-acetylsthioacetate (SATA) solution to the above VB. 12 In BSA, the reaction was carried out at room temperature for 30 minutes, followed by desalting and purification using a desalting column. The eluent was concentrated to 2 mg / mL. The activated VB was then... 12 Mix BSA and AP solutions, react overnight at 2-8°C, add an equal volume of glycerol, and store at -20°C. Add VB... 12 -BSA-labeled alkaline phosphatase markers were diluted 1000-fold with 50 mmol / L LMES (pH 5.0) buffer to obtain VB. 12 -BSA-labeled alkaline phosphatase working solution (1 μg / mL).

[0049] 4) Cleaning solution: Weigh 2.383 g of HEPES and dissolve it in purified water. Adjust the pH to around 7.0 using sodium hydroxide. Add 1 mL of Tween 20 and 0.5 g of sodium azide, stir to mix well, and bring the volume to 1 L.

[0050] 5) Substrate solution: Commercially available APS-5 substrate solution (purchased from Dorest (Shenzhen) Medical Technology Co., Ltd.). Example

[0051] This embodiment provides a vitamin B 12 The detection method is based on the detection kit provided in Example 3, and the specific steps are as follows: 1) Mix 50 μL of the sample to be tested and 25 μL of the sample processing solution at a volume ratio of 2:1, and react at 37℃ for 10 minutes; 2) Add 50 μL of biotin working solution labeled with internal factor to the reaction system, mix well, and react at 37°C for 5 minutes; 3) Add the reaction mixture from step 2) to 50 μL of VB. 12 -BSA-labeled alkaline phosphatase working solution was added to the magnetic bead reagent and mixed well, and reacted at 37°C for 5 minutes; 4) After the reaction is complete, perform magnetic separation and wash three times with cleaning solution; 5) After adding 100 μL of substrate solution and incubating for 2 minutes, the photoelectric signal was then interpreted.

[0052] This experiment tested the effects of sample treatment solutions containing different concentrations of sodium hydroxide and sodium lauryl sarcosinate on vitamin B1. 12 The effect of the detection effect was also tested on the vitamin B provided in Example 4. 12 The linearity, accuracy, and repeatability of the detection method were evaluated, and the results were compared with those of commercially available detection kits.

[0053] 1. Determination of the optimal formulation of the sample processing solution: To determine the optimal sample processing solution formulation, 0.5%, 2%, 5%, and 10% sodium hydroxide solutions were prepared, and 0.2%, 0.5%, 2%, and 10% sodium dodecyl sarcosinate were added to them respectively. The optimal component concentration was selected by testing the same batch of serum samples (from the Third People's Hospital of Shenzhen) and based on the correlation between the test results and theoretical values.

[0054] The 16 different concentration formulations are shown in Table 1. The clinical samples used in the tests contained vitamin B... 12 The theoretical values ​​and the test values ​​obtained from 16 sample treatment solutions are shown in Tables 2-17. The detection method used in the test was the same as described in Example 4, only the formulation of the sample treatment solution was replaced, and the steps were kept the same.

[0055] Table 1. Sample treatment solution formulations at different concentrations

[0056] Table 2 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to Formula 1

[0057] Table 3 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to Formula 2

[0058] Table 4 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 3

[0059] Table 5 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 4

[0060] Table 6 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 5

[0061] Table 7 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 6

[0062] Table 8 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 7

[0063] Table 9 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 8

[0064] Table 10 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 9

[0065] Table 11 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 10

[0066] Table 12 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 11

[0067] Table 13 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 12

[0068] Table 14 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 13

[0069] Table 15 Vitamin B in clinical samples 12 The theoretical value and the test value corresponding to formula 14

[0070] Table 16 Vitamin B in Clinical Samples 12 The theoretical value and the test value corresponding to formula 15

[0071] Table 17 Vitamin B in clinical samples 12The theoretical value and the test value corresponding to formula 16

[0072] The results in Tables 2 to 17 show that Formula 2, Formula 6, and Formula 10 have good clinical relevance. Formula 2 has the best correlation coefficient (R) greater than 0.975, and is therefore the optimal formula, which is 2% sodium hydroxide and 0.2% sodium dodecyl sarcosinate.

[0073] 2. Linearity verification: The 2000 pg / mL sample was diluted to five concentration gradients at a certain ratio, with the lowest concentration sample being close to the lower limit of the linear range. Each concentration sample was tested three times using the detection method provided in Example 4, and the average value was calculated. The average value and the dilution ratio were then fitted with a linear regression line using the least squares method to calculate the correlation coefficient. The results are shown in Table 18.

[0074] Table 18 Linearity Validation Results (Unit: pg / mL)

[0075] As can be seen from the data in Table 18, the vitamin B provided in Example 4 of this invention... 12 The detection method yielded good linearity, with a linear correlation coefficient R of 0.999 and a detection limit of 100 pg / mL.

[0076] 3. Accuracy Test: Using VB 12 The international reference material was used as the test sample (reconstituted with purified water) and tested according to the test method provided in Example 4. Each sample was measured three times, and the test result was recorded as xi. The relative deviation was calculated using the following formula: Where B refers to relative deviation, xi refers to measured concentration, and T refers to the index of fixed concentration.

[0077] The results are shown in Table 19.

[0078] Table 19 Accuracy test data (unit: pg / mL)

[0079] As can be seen from the data in Table 19, the detection method provided in Embodiment 4 of the present invention is effective in detecting VB. 12 The accuracy deviation of the international reference material is within 6%, which meets the testing performance requirements.

[0080] 4. Repeatability testing: The test method provided in Example 4 was used to test clinical serum samples with known concentrations of ≤250 pg / mL, 400~700 pg / mL, and 1000~1500 pg / mL, with each test repeated 10 times. The average value of the 10 measurements was calculated. The coefficient of variation (CV) is calculated using the following formula, which is based on the standard deviation (SD) and the variability (CV).

[0081] ;in, The mean of the measurement results is denoted by SD, the standard deviation is SD, and the coefficient of variation is CV. The results are shown in Table 20.

[0082] Table 20 Repeatability Test Results

[0083] As can be seen from the data in Table 20, the coefficient of variation of the detection method provided in Embodiment 4 of the present invention is within 7%, which meets the performance requirements.

[0084] 5. Methodological Comparison: The detection method provided in Example 4 was used in conjunction with Roche Diagnostics' vitamin B12 assay. 12 The detection kit (electrochemiluminescence immunoassay, catalog number: 04745736190) was used to test 100 serum samples (from the Third People's Hospital of Shenzhen). The correlation between the two was compared, and the results are shown in Table 21. A scatter plot was then created using the data from Table 21. Figure 1 As shown.

[0085] Table 21 Methodological Comparison Results

[0086] From Table 21 and Figure 1 The results show that the kit provided in Example 3 of this invention / the detection method provided in Example 4 have good correlation with the commercial Roche test kit when detecting clinical samples, with a correlation coefficient R. 2 It is 0.9539.

[0087] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A sample processing solution, characterized in that, The sample processing solution includes a strong alkali and sodium dodecyl sarcosinate.

2. The sample processing solution according to claim 1, characterized in that, The sample processing solution comprises 5-100 mg / mL strong alkali and 1-20 mg / mL sodium dodecyl sarcosinate.

3. A type of vitamin B 12 The sample pretreatment method for detection is characterized by, The sample pretreatment method includes: mixing and reacting the sample processing solution according to claim 1 or 2 with the sample to be tested.

4. The vitamin B according to claim 3 12 The sample pretreatment method for detection is characterized by, The volume ratio of the sample processing solution to the sample to be tested is 1:(1~8). Preferably, the reaction time is 5 to 30 minutes; Preferably, the reaction temperature is 34℃~40℃.

5. A type of vitamin B 12 The test kit is characterized by, The vitamin B 12 The test kit comprises: the sample processing solution as described in claim 1 or 2, streptavidin-labeled magnetic beads, intrinsic factor-labeled biotin, labeled enzyme, and chemiluminescent substrate solution.

6. The vitamin B according to claim 5 12 The test kit is characterized by, The labeling enzyme includes vitamin B. 12 Derivative-labeled alkaline phosphatase; Preferably, the chemiluminescent substrate solution includes AMPPD or APS-5.

7. The vitamin B according to claim 5 12 The test kit is characterized by, The mass ratio of streptavidin to magnetic beads in the streptavidin-labeled magnetic beads is 1:(40~60). Preferably, the molar ratio of intrinsic factor to biotin in the intrinsic factor-labeled biotin is 1:(10~80).

8. A type of vitamin B 12 The detection method is characterized by, The detection method uses vitamin B as described in any one of claims 5 to 7. 12 The test kit performs the test, which includes the following steps: S1: Mix the sample to be tested with the sample processing solution at a volume ratio of (1~8):1 and react thoroughly. S2: Add biotin labeled with intrinsic factor to the reaction system in step S1 and mix well for reaction; S3: Add streptavidin-labeled magnetic beads and labeled enzyme to the reaction system in step S2 and mix well. S4: Perform magnetic separation, wash with cleaning solution 2-4 times, add chemiluminescent substrate solution, and then perform photoelectric signal interpretation.

9. The detection method according to claim 8, characterized in that, The reaction time in step S1 is 5~30 min, and the reaction temperature is 34℃~40℃; Preferably, the reaction time in steps S2 and S3 is 3~8 min and the reaction temperature is 34℃~40℃.

10. The sample processing solution according to claim 1 or 2 for detecting vitamin B in biological samples. 12 Applications in [the field].

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