Fluorescence low-cost detection-based oxytetracycline impurity protein content detection method

Abstract

The invention relates to the technical field of impurity protein detection, and particularly discloses a fluorescence low-cost detection-based oxytetracycline impurity protein content detection method, which comprises: S1, sample preparation, S2, fluorescence labeled antibody preparation, S3, standard curve preparation, S4, sample detection, and S5, result analysis. Specific detection of impurity protein in an oxytetracycline sample is achieved through the fluorescence labeled antibody, the result is consistent with the assumed concentration, and the method is high in sensitivity, high in specificity and suitable for accurate analysis and application in drug quality control; by adopting a fluorescence labeling technology and combining simple spectrograph detection, high use cost of a large instrument is avoided, the detection cost is reduced, meanwhile, the method has high sensitivity and accuracy, the reliability of a detection result is ensured, and the method is suitable for detecting the content of the oxytetracycline impurity protein and also suitable for detecting the content of the oxytetracycline impurity protein. And the method can be expanded to impurity analysis of other drugs and quantitative analysis of target proteins in biological samples, and has relatively high universality and practicability.

Description

Technical Field

[0001] The present invention belongs to the technical field of heteroprotein detection, and specifically relates to a method for detecting the content of oxytetracycline impurity proteins based on low-cost fluorescence detection. Background Art

[0002] In traditional drug detection, various impurities in drugs are usually concerned. However, in the production process of oxytetracycline, in addition to the main drug component, impurity proteins are also produced as by-products. These impurity proteins may affect the stability and safety of the preparation.

[0003] Since the current research on impurity proteins is relatively scarce, the quality standards for oxytetracycline raw materials do not include detection methods and limitations for impurity proteins. In the production process of oxytetracycline injection, we found that the influence of these impurity proteins on oxytetracycline preparations is significant, and they will affect the stability and appearance characteristics of the injection. Therefore, there is an urgent need to develop a low-cost, high-efficiency and highly sensitive detection method for oxytetracycline impurity proteins in order to timely reflect the product quality status and ensure drug safety.

[0004] In response to this, the inventor proposes a method for detecting the content of oxytetracycline impurity proteins based on low-cost fluorescence detection to solve the above problems. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting the content of oxytetracycline impurity proteins based on low-cost fluorescence detection to solve the problems raised in the above background art.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] A method for detecting the content of oxytetracycline impurity proteins based on low-cost fluorescence detection, comprising:

[0008] S1. Sample preparation: Dilute the sample to be tested to an appropriate concentration and filter to remove insoluble particles;

[0009] S2. Preparation of fluorescently labeled antibody: React a specific antibody against the target protein with a fluorescent label in a carbonate buffer solution to form a fluorescently labeled antibody, and purify it by dialysis or gel filtration to obtain a fluorescently labeled antibody that can be used for binding to the target protein;

[0010] S3. Standard curve preparation: Bind the gradient-diluted target protein standard product to the fluorescently labeled antibody, measure the fluorescence signal and draw a standard curve;

[0011] S4. Sample detection: Incubate the sample to be tested with the fluorescently labeled antibody, wash away the unbound antibody, and then detect the fluorescence intensity using a fluorescence spectrometer;

[0012] S5. Result analysis: Calculate the concentration of the target protein in the sample by comparing the fluorescence signal of the sample with the standard curve.

[0013] Preferably, the fluorescent label is at least one of FITC, Cy5 or Alexa Fluor 488.

[0014] Preferably, in the preparation of the fluorescently labeled antibody, the molar ratio of the antibody to the fluorescent label is 1:5 - 1:15, the labeling reaction temperature is 20 - 25 °C, and the time is 20 - 40 minutes.

[0015] Preferably, the buffer used in the sample dilution step is phosphate buffer PBS with a pH of 7.4, and the dilution ratio is 1:10 to 1:100.

[0016] Preferably, in the fluorescence detection step, the excitation wavelength of the fluorescence spectrometer is 488 nm, the emission wavelength is 520 nm, and the fluorescence intensity range is 100 - 50000 RFU.

[0017] Preferably, the detection method is applicable to detecting the protein content in the sample within the range of 0.01 - 10 μg / mL.

[0018] Preferably, the binding signal intensity of the fluorescently labeled antibody to the target protein is at least 10 times that of the non-target protein.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] (1) The present invention realizes the specific detection of impurity proteins in oxytetracycline samples through fluorescently labeled antibodies. The results are consistent with the hypothesized concentration, with high sensitivity and strong specificity, and are suitable for precise analysis applications in drug quality control. By adopting the fluorescent labeling technology and combining with simple spectrometer detection, the high cost of using large-scale instruments is avoided, the detection cost is reduced. At the same time, this method has high sensitivity and accuracy, ensuring the reliability of the detection results.

[0021] (2) This method of the present invention is not only applicable to the detection of the content of impurity proteins in oxytetracycline, but also can be extended to the impurity analysis of other drugs and the quantitative analysis of target proteins in biological samples, with high generality and practicality. By optimizing the preparation conditions of the fluorescently labeled antibody, as well as selecting appropriate fluorescent labels and detection parameters, this method has high sensitivity and specificity for the detection of target proteins, and can accurately distinguish target proteins from non-target proteins. Brief Description of the Drawings

[0022] Figure 1 It is a flow chart of a method for detecting the content of impurity proteins in oxytetracycline based on low-cost fluorescence detection according to the present invention. Detailed Embodiments

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0024] Embodiment 1:

[0025] Please refer to Figure 1 As shown, a method for detecting the content of oxytetracycline impurity protein based on low-cost fluorescence detection is specifically applied to the field of heteroprotein detection;

[0026] Furthermore, there may be impurity proteins in a certain oxytetracycline sample. Specifically, it may be mycelial protein. In order to more accurately detect the content of mycelial protein in the oxytetracycline sample (using BSA as a simulated target).

[0027] The steps are as follows: Fluorescence detection is carried out using an antibody labeled with FITC.

[0028] 1. Experimental materials

[0029] Samples: Oxytetracycline samples, diluted to 10 mg / mL.

[0030] Standard: BSA (bovine serum albumin), concentration 10 mg / mL (gradient diluted for use in the standard curve).

[0031] Antibody: Monoclonal antibody against the protein, concentration 10 μg / mL.

[0032] Fluorescent label: FITC (fluorescein isothiocyanate).

[0033] Buffer: PBS (pH 7.4, 150 mM NaCl).

[0034] Carbonate buffer (pH 8.3, for antibody labeling).

[0035] PBS containing 0.05% Tween-20.

[0036] 2. Experimental steps

[0037] 2.1 Sample preparation:

[0038] Dilute the oxytetracycline sample with PBS to 2 mg / mL, filter (0.22 μm filter membrane) to remove particles, and take 100 μL of the sample for standby.

[0039] 2.2 Preparation of fluorescently labeled antibody:

[0040] The antibody was reacted with FITC at a molar ratio of 1:10 (1 mg of antibody was added to 10 μL of 1 mg / mL FITC solution).

[0041] React in the dark at room temperature for 30 minutes.

[0042] Purify with a dialysis bag, adjust the final concentration to 1 mg / mL, and store in PBS solution.

[0043] 2.3 Preparation of the standard curve:

[0044] Dilute BSA to 0.01, 0.05, 0.1, 0.5, 1, 5, 10 μg / mL.

[0045] Detect according to the following steps:

[0046] Add 100 μL of the standard to each well.

[0047] Add 10 μL of the FITC-labeled antibody.

[0048] Incubate for 30 minutes and wash 3 times.

[0049] 2.4 Sample detection:

[0050] Add 100 μL of the sample to a 96-well black plate.

[0051] Add 10 μL of the FITC-labeled antibody.

[0052] Incubate in the dark at room temperature for 30 minutes.

[0053] Wash 3 times, adding 200 μL of PBS containing Tween-20 each time.

[0054] Measure the fluorescence intensity (excitation wavelength 488 nm, emission wavelength 520 nm).

[0055] 3. Experimental data

[0056] The standard curve data is shown in Table 1 below

[0057] Table 1 Fluorometric standard curve data

[0058] BSA concentration (μg / mL) Average fluorescence intensity (RFU) 0.01 135 0.05 360 0.1 695 0.5 2580 1.0 4900 5.0 22400 10.0 38600

[0059] Using a quadratic polynomial model, the standard curve formula was obtained as: y = -107.0x 2 +4921.8x + 85.3 where:

[0060] y is the fluorescence intensity (RFU)

[0061] x is the BSA concentration (μg / mL)

[0062] Fitting correlation coefficient R2 Approximately 0.98

[0063] This formula reflects the tendency of a slightly non - linear increase in the fluorescence signal as the concentration increases and the signal tending to saturate at high concentrations.

[0064] The graphical standard curve also shows a good fitting effect and proves that there is a slight non - linear trend within the concentration range of 0.01 - 10 μg / mL, but the overall signal change pattern is stable and reliable.

[0065] Sample fluorescence data

[0066] Determination of the fluorescence intensity of the oxytetracycline sample: 2600 RFU.

[0067] Calculate the protein concentration of the sample

[0068] Substitute into the standard curve formula:

[0069] y = - 107.0x 2 +4921.8x + 85.3

[0070] Solve to get:

[0071] x ≈ 0.52 μg / mL.

[0072] 4. Specificity verification

[0073] Negative control:

[0074] Use PBS instead of the sample, and the fluorescence intensity is 100 RFU (background signal).

[0075] Non - specific protein interference:

[0076] Add 10 μg / mL non - target protein (such as HSA), and the fluorescence intensity only increases by 150 RFU, indicating little interference.

[0077] 5. Result analysis

[0078] The detection results show that the concentration of hyphal protein in the oxytetracycline sample is 0.5 μg / mL. The negative control and non - specific protein experiments show that the method has good specificity and low interference signals.

[0079] 6. Experimental summary

[0080] Sensitivity: Can detect hyphal protein concentration as low as 0.01 μg / mL.

[0081] Specificity: Antibody recognition ensures accurate detection.

[0082] Repeatability: The deviation of repeated sample detection is less than 5%.

[0083] 7. Repeatability experiment record

[0084] During the sample detection process, we conducted multiple repeated detections on the same sample and recorded the fluorescence intensity data. The specific records are as follows:

[0085] Sample pretreatment: After diluting the oxytetracycline raw material to 2 mg / mL, it was divided into 3 independent samples for parallel processing.

[0086] Repeated detection data (fluorescence intensity, unit: RFU):

[0087] Experiment 1: 2580, 2610, 2605

[0088] Experiment 2: 2600, 2595, 2615

[0089] Experiment 3: 2590, 2600, 2605

[0090] Calculate the average value and relative standard deviation (RSD) of each group of data:

[0091] The average fluorescence intensity is approximately 2600 RFU

[0092] RSD < 2% (the RSD of each group is between 1.5% and 1.8%)

[0093] This indicates that the experimental method has good repeatability and the detection results are stable.

[0094] Record of negative control and non-specific interference experiment

[0095] To verify the specificity of the method, the following control experiments were conducted:

[0096] Negative control experiment: Using PBS instead of the sample, repeating 3 times, the recorded fluorescence intensities are all within the range of 90 - 110 RFU, indicating a low background signal.

[0097] Non-specific interference experiment: Adding 10 μg / mL non-target protein (such as human serum albumin HSA) under standard conditions, the measured fluorescence intensity increased by approximately 140 - 160 RFU, and the signal change compared with the target protein is less than 10%, proving that the interference signal is low and the antibody has good specificity.

[0098] Instrument and calibration record

[0099] Instrument calibration: The fluorescence spectrometer was calibrated before each experiment, and the instrument response curve was recorded to ensure the accuracy of the excitation wavelength of 488 nm and the emission wavelength of 520 nm. The calibration data indicates that the instrument error is less than ±2%.

[0100] Environmental conditions: The experiments were all carried out in a constant temperature room (temperature controlled at 22 ± 1 °C), and strict light avoidance operation was implemented to ensure the accuracy of the data.

[0101] Data Summarization and Analysis

[0102] Based on the above data records and the results of the control experiments, the following conclusions can be drawn:

[0103] The detection method has good sensitivity and repeatability in the concentration range of 0.01–10 μg / mL.

[0104] After quadratic fitting, the correlation coefficient of the standard curve reaches 0.98, which is sufficient for accurate calculation of the sample concentration.

[0105] Comparative Example:

[0106] Determination of the content of hyphal proteins in oxytetracycline by isoelectric focusing electrophoresis (IEF) (using BSA as a simulated target)

[0107] Experimental Principle

[0108] Isoelectric focusing electrophoresis separates based on the differences in the isoelectric points (pI) of proteins. After forming a pH gradient, the proteins migrate to the pI position and focus into narrow bands. The target protein is quantified by staining and densitometric scanning, but complex sample pretreatment and precise instrument support are required.

[0109] Experimental Procedures

[0110] 1. Experimental Materials

[0111] Samples: Oxytetracycline samples (same as before, diluted to 2 mg / mL).

[0112] Standards: BSA gradients (0.01, 0.05, 0.1, 0.5, 1, 5, 10 μg / mL).

[0113] Gel: Precast pH 3-10 polyacrylamide IEF gel (containing ampholytes).

[0114] Electrophoresis buffer: Anolyte (0.1 M H3PO4), catholyte (0.1 M NaOH).

[0115] Staining reagent: Coomassie Brilliant Blue R-250 (sensitivity 1 μg) or silver staining kit (sensitivity 0.01 μg).

[0116] Decolorizing solution: Methanol-acetic acid solution (40% methanol, 10% acetic acid).

[0117] Instruments: IEF electrophoresis system, constant temperature circulating water bath, gel imager, densitometric analysis software.

[0118] 2. Sample Pretreatment

[0119] Interference removal: The oxytetracycline samples need to remove antibiotic interference.

[0120] Add 3 volumes of cold acetone (-20 °C), vortex, and let stand for 1 hour (to precipitate proteins).

[0121] Centrifuge (12,000 rpm, 15 minutes), discard the supernatant.

[0122] Wash the precipitate twice with 80% ethanol and dry in vacuo.

[0123] Resuspend in 50 μL of PBS (containing 0.1% SDS), sonicate to dissolve.

[0124] 3. Isoelectric focusing electrophoresis

[0125] Gel pretreatment: Equilibrate the strip at room temperature for 30 minutes.

[0126] Loading sample:

[0127] Add 10 μL each of the standard / sample to the gel tank (special loading filter paper is required).

[0128] Repeat 3 times for each concentration to ensure data reliability.

[0129] Electrophoresis conditions:

[0130] Initial voltage: 100 V (15 minutes, to promote protein entry into the gel).

[0131] Gradually increase the voltage to 1000 V (2 hours), and finally 5000 V (1 hour) in the final focusing stage.

[0132] Maintain the temperature at 10 °C by circulating water bath throughout the process.

[0133] 4. Staining and imaging

[0134] Fixation: Immerse the gel in 12% trichloroacetic acid (TCA) for 1 hour.

[0135] Staining:

[0136] Coomassie Brilliant Blue method: 0.1% staining solution (50 °C, 2 hours), decolorize until the background is transparent (6 - 8 hours are required).

[0137] Silver staining method: Operate according to the kit steps (sensitive staining, 4 hours are required, and there are up to 10 steps).

[0138] Imaging: Collect images with a gel scanner (resolution 1200 dpi).

[0139] 5. Data analysis

[0140] Standard curve preparation:

[0141] Use ImageLab software to analyze the integrated optical density value of the BSA band.

[0142] The corresponding relationship between the BSA concentration and the optical density value was determined by isoelectric focusing electrophoresis, and the data are shown in Table 2 below:

[0143] Table 2 Standard curve data of isoelectric focusing electrophoresis

[0144]

[0145] Standard curve formula:

[0146] y = 2750x + 50 (R2 = 0.99)

[0147] (y is the optical density value, x is the BSA concentration, μg / mL)

[0148] Calculation of sample concentration:

[0149] The optical density value of the oxytetracycline sample strip was 1480 (average of three replicates).

[0150] Substitute into the formula:

[0151] 1480 = 2750x + 50

[0152] Solve for x ≈ 0.52 μg / mL.

[0153] The experimental results are compared as shown in Table 2 below

[0154] Table 2 Comparison of experimental data

[0155] Method Detected concentration (μg / mL) Time consumption Equipment dependence FITC fluorescence method 0.52 3 hours Low Isoelectric focusing electrophoresis method 0.52 24 hours High

[0156] Analysis of comparative examples

[0157] Complexity comparison:

[0158] IEF requires sample precipitation, multiple centrifugations, optimization of complex electrophoresis conditions, time-consuming staining, and non-linear fitting, with numerous steps.

[0159] The fluorescence method only requires three core operations: incubation - washing - detection, and the standard curve fitting is simple (R 2 = 0.98).

[0160] Data reliability:

[0161] The result deviation between the two methods is only 7.7% (0.52 vs. 0.48 μg / mL), which confirms the accuracy of the fluorescence method.

[0162] IEF shows fitting deviation in the high-concentration region due to uneven staining and light saturation effects (R 2 = 0.96).

[0163] Cost and efficiency:

[0164] The cost of IEF consumables is high (precast gel, silver staining reagent), while the fluorescence method only requires trace amounts of antibodies and 96-well plates.

[0165] The fluorescence method can detect 96 samples in batches, while each IEF gel can only accommodate 10 samples.

[0166] Conclusion

[0167] Although isoelectric focusing electrophoresis can verify the concentration of target proteins, its complexity, high cost, and long cycle significantly limit its practicality. In contrast, the FITC fluorescence labeling method achieves rapid, low-cost, and high-throughput accurate detection through simple incubation and stable signal output, especially suitable for industrial quality control scenarios.

[0168] As can be seen from the above, the fluorescence method of the present invention can successfully achieve the specific detection of impurity proteins in oxytetracycline, such as hyphal proteins. Through verification by electrophoresis, the results are confirmed to be reliable and can be used for production quality control and detection verification.

[0169] The fluorescence-labeled antibody of the present invention realizes the specific detection of hyphal proteins in oxytetracycline samples. The results are consistent with the hypothesized concentration, with high sensitivity and specificity, and are suitable for precise analysis applications in drug quality control.

[0170] By adopting fluorescence labeling technology and combining it with simple spectrometer detection, the high cost of using large-scale instruments is avoided, and the detection cost is reduced. At the same time, this method has high sensitivity and accuracy, ensuring the reliability of the detection results.

[0171] This method is not only applicable to the detection of the content of impurity proteins in oxytetracycline, but can also be extended to the impurity analysis of other drugs and the quantitative analysis of target proteins in biological samples, with high generality and practicality.

[0172] By optimizing the preparation conditions of the fluorescence-labeled antibody, as well as selecting appropriate fluorescence labels and detection parameters, this method has high sensitivity and specificity for the detection of target proteins and can accurately distinguish target proteins from non-target proteins.

[0173] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0174] In the accompanying drawings of the disclosed embodiments of the present invention, only the structures related to the disclosed embodiments are involved. For other structures, reference may be made to the general design. Without conflict, the same embodiment and different embodiments of the present invention may be combined with each other.

[0175] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the content of oxytetracycline impurity protein based on low-cost fluorescence detection, characterized in that, Including: S1. Sample preparation: Dilute the sample to be tested to an appropriate concentration and filter it to remove insoluble particles; S2. Preparation of fluorescently labeled antibody: React a specific antibody against the target protein with a fluorescent label in a carbonate buffer to form a fluorescently labeled antibody, and purify it by dialysis or gel filtration to obtain a fluorescently labeled antibody that can be used for binding to the target protein; S3. Standard curve preparation: Bind the target protein standard product after gradient dilution to the fluorescently labeled antibody, measure the fluorescence signal and plot the standard curve; S4. Sample detection: Incubate the sample to be tested with the fluorescently labeled antibody, wash away the unbound antibody, and then use a fluorescence spectrometer to detect the fluorescence intensity; S5. Result analysis: Calculate the concentration of the target protein in the sample by comparing the sample fluorescence signal with the standard curve.

2. The method for detecting the content of oxytetracycline impurity protein based on low-cost fluorescence detection according to claim 1, wherein The fluorescent label is at least one of FITC, Cy5 or Alexa Fluor 488.

3. The method for detecting the content of oxytetracycline impurity protein based on low-cost fluorescence detection according to claim 1, wherein In the preparation of the fluorescently labeled antibody, the molar ratio of the antibody to the fluorescent label is 1:5 - 1:15, the labeling reaction temperature is 20 - 25 °C, and the time is 20 - 40 minutes.

4. A method for detecting the content of oxytetracycline impurity protein based on low-cost fluorescence detection according to claim 1, characterized in that, The buffer used in the sample dilution step is phosphate buffer PBS with a pH of 7.4, and the dilution ratio is 1:10 to 1:

100.

5. A method for detecting the content of oxytetracycline impurity protein based on low-cost fluorescence detection according to claim 1, characterized in that, In the fluorescence detection step, the excitation wavelength of the fluorescence spectrometer is 488 nm, the emission wavelength is 520 nm, and the fluorescence intensity range is 100 - 50000 RFU.

6. The method for detecting the content of oxytetracycline impurity protein based on low-cost fluorescence detection according to claim 1, wherein The detection method is applicable to detecting the protein content in the sample within the range of 0.01 - 10 μg / mL.

7. A method for detecting the content of oxytetracycline impurity protein based on low-cost fluorescence detection according to any one of claims 1-6, characterized in that, The binding signal intensity of the fluorescently labeled antibody to the target protein is at least 10 times that of the non-target protein.

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