A method for detecting the efficiency of biotin labeling of proteins

Abstract

The application provides a method for detecting the labeling efficiency of protein biotin, characterized in that: after denaturation of the protein, the protein is incubated with blocked magnetic beads, and the labeling efficiency of the biotin protein is calculated through the concentration change of the biotin protein before and after incubation with the magnetic beads. The scheme has good stability and high accuracy.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more specifically, to a method for detecting the efficiency of protein biotin labeling. Background Technology

[0002] Currently, commonly used biotin quantitative assay kits on the market produce unstable results. Their detection principle is based on the absorbance change of HABA (4'-hydroxyazobenzene-2-carboxylic acid). However, this method is not suitable for the labeling efficiency of biotin-labeled proteins due to instability, poor repeatability, and variations in results between different batches and at different times. This means that existing methods cannot provide stable and reliable quality control for biotin-labeled protein products, which will seriously affect the consistency and accuracy of downstream applications such as immunoassays.

[0003] The reasons for these defects may be as follows:

[0004] 1. Susceptible to interference: The HABA method relies on the accurate measurement of absorbance. If there are other impurities in the sample that absorb near 500nm, they will seriously interfere with the results.

[0005] 2. Unable to detect sterically hindered biotin: When biotin is labeled inside a protein or is obscured by the protein's higher-order structure, HABA cannot effectively compete for binding, resulting in a measured labeling efficiency lower than the actual value.

[0006] 3. When the same protein sample is tested using different brands, different batches, or at different time points using the HABA method, the coefficient of variation (CV%) is too high, which fails to meet the quality control requirements. Summary of the Invention

[0007] The present invention aims to overcome the above-mentioned defects and provide a method for detecting the efficiency of protein biotin labeling with good stability and strong anti-interference ability.

[0008] The present invention provides a method for detecting the biotin labeling efficiency of a protein, namely, after denaturing the protein, incubating it with sealed magnetic beads, and calculating the biotin labeling efficiency by measuring the concentration change of biotin before and after incubation with the magnetic beads.

[0009] Furthermore, the method for detecting protein biotin labeling efficiency provided by the present invention also has the characteristic that the denaturation method is selected from at least one of the following methods A and D:

[0010] Method A. Add a chemical denaturant;

[0011] Method B. Add ionic or nonionic detergents;

[0012] Method C. Physical denaturation;

[0013] Method D. Add a reducing agent.

[0014] Furthermore, the method for detecting protein biotin labeling efficiency provided by the present invention also has the following characteristics: the magnetic bead sealing method includes sealing the magnetic beads using an inert protein.

[0015] Furthermore, the method for detecting the biotin labeling efficiency of a protein provided by the present invention also has the following feature: the magnetic bead blocking method further includes a second blocking of the magnetic beads using a homologous non-biotin of the protein to be tested.

[0016] Furthermore, the method for detecting protein biotin labeling efficiency provided by the present invention also has the following characteristics, namely, it includes the following steps:

[0017] Step 1. According to the sample label concentration, dilute the non-biotinylated protein and biotinylated protein to the specified concentration with denaturing agent, and denature overnight at low temperature;

[0018] Step 2. Detect the concentrations of non-biotinylated protein and biotinylated protein;

[0019] Step 3. Use protein or polymer to seal the magnetic beads for the first time;

[0020] Step 4. Perform a second blocking of the magnetic beads using non-biotinylated protein;

[0021] Step 5. Bind the biotinylated protein to the magnetic beads and obtain the concentration values ​​of the biotinylated protein before and after binding to the magnetic beads;

[0022] Step 6. Calculate biotin labeling efficiency.

[0023] Furthermore, the method for detecting the biotin labeling efficiency of proteins provided by the present invention also has the following characteristics: the biotin labeling efficiency is calculated as the concentration difference of biotinylated protein before and after binding to the magnetic beads / the initial biotinylated protein concentration / 2, which is the biotin labeling efficiency.

[0024] Furthermore, the method for detecting the biotin labeling efficiency of proteins provided by the present invention also has the following characteristics: in step 4, the magnetic beads that have been first blocked are resuspended in non-biotinylated protein and incubated at room temperature for 1-2 hours on a constant temperature shaker before magnetic separation.

[0025] Furthermore, the method for detecting the biotin labeling efficiency of proteins provided by the present invention also has the following characteristics: in step 5, the biotinylated protein is resuspended in the magnetic beads that have been sealed for the second time, and then magnetic separation is performed after incubation at room temperature on a constant temperature shaker for 1-2 hours.

[0026] In addition, the present invention provides a product for detecting the biotin labeling efficiency of proteins, comprising biotin-labeled proteins, non-biotin-labeled proteins, proteins or polymers, streptavidin-magnetic beads, and denaturing buffers.

[0027] Furthermore, the present invention also provides a system for detecting the efficiency of protein biotin labeling, comprising means for sequentially performing the steps in the above-described method.

[0028] The function and effects of this invention:

[0029] This invention uses streptavidin magnetic beads for detection. The labeling efficiency of biotinylated proteins is calculated by measuring the concentration change before and after incubation with magnetic beads after denaturing the biotinylated protein to fully expose the biotin groups. The streptavidin-biotin (SA-Biotin) system has extremely high binding affinity (Kd=10). -15 Therefore, this method is very stable. The consistency and stability of the detection results have been verified through testing on a large number of samples.

[0030] First, this invention achieves result verification through a direct quantitative method, thus ensuring stable results. Specifically, this invention abandons the indirect competitive substitution principle and directly utilizes streptavidin magnetic beads to capture biotinylated proteins. The labeling efficiency is calculated by measuring the difference (Δ value) in protein concentration in the supernatant before and after the reaction. This method is based on the unparalleled high affinity of SA-Biotin, resulting in a robust and specific binding process that is minimally affected by environmental factors, thus yielding extremely stable results (CV% < 5%).

[0031] Secondly, this invention introduces denaturation conditions to make the detection results more accurate. Specifically, this invention creatively performs the binding reaction under denaturation conditions. Denaturing agents (such as urea and SDS) can open up the protein's spatial structure, fully exposing all labeled biotin sites and avoiding "false negatives" caused by protein folding, thereby detecting the true total biotin labeling efficiency. At the same time, this condition can also effectively reduce non-specific binding caused by hydrophobic and electrostatic interactions, thereby reducing false positive signals and background noise. This "two-pronged" effect—simultaneously improving the sensitivity and specificity of detection—jointly ensures the ultra-high accuracy of the final labeling efficiency calculation result, which cannot be achieved by traditional detection methods performed under natural conditions (such as the HABA method).

[0032] Thirdly, this invention improves accuracy through a unique blocking strategy. Specifically, this invention designs a two-step blocking method (first using BSA to block non-specific sites on magnetic beads, and then using homologous non-biotinylated proteins for competitive blocking), which minimizes non-specific adsorption, ensures the accuracy of concentration measurements (M1, M2), and makes the final calculated labeling efficiency highly reliable. Detailed Implementation

[0033] This invention can be implemented in many ways and can have various embodiments, but this is not intended to limit the invention to a specific implementation method, but should be understood to include all modifications, equivalents and even substitutes that fall within the spirit and technical scope of this invention.

[0034] Example 1. A method for detecting the efficiency of protein biotin labeling.

[0035] This embodiment aims to establish a testing method for the biotin labeling efficiency of proteins and standardize operational requirements to ensure the accuracy of test results, thereby enabling the testing of biotin labeling efficiency of protein products.

[0036] In this embodiment, based on the high affinity (Kd = 1 × 10⁻¹⁵ M) between biotin and streptavidin, biotin-labeled proteins were incubated with SA magnetic beads. The supernatant was then collected, containing only unlabeled protein. The protein exhibited light absorption at 280 nm, which was detected using Nanodrop, and the protein concentration was calculated. The concentration of the protein solution before and after incubation was measured, and the difference divided by the initial protein concentration represents the biotin labeling efficiency.

[0037] The specific method is as follows:

[0038] 1. Key experimental materials and equipment 2.

[0040] 3. Experimental Methods

[0041] I. Sample denaturation and concentration determination

[0042] SI1. Based on the sample labeling concentration, dilute the non-biotinylated protein to 0.69 mg / ml, 100 μl; dilute the biotinylated protein to 0.23 mg / ml, 80 μl, and denature at 4-37 degrees Celsius for 0.1-16 hours.

[0043] Regarding sex reassignment, any one or a combination of the following methods may be used:

[0044] If using denaturing agents, chemical denaturants such as urea, guanidine hydrochloride, sodium hydroxide, sodium chloride, magnesium chloride, and arginine can be used; nonionic detergents such as Tween 20, Triton X-100, and NP-40 can be used; ionic detergents such as SDS can be used; physical denaturing conditions such as high temperature, high pressure, mechanical force, and extreme pH can also be used; and reducing agents such as β-mercaptoethanol (BME), dithiothreitol (DTT), and tris(2-carboxyethyl)phosphine (TCEP) can also be used for denaturation.

[0045] It is worth noting that conventional thorough denaturation methods, such as the most typical example of SDS-PAGE (protein gel electrophoresis) sample processing, use high-intensity chemical denaturants and high-temperature boiling to completely and irreversibly destroy the protein structure, even at the cost of sacrificing all protein functions to achieve denaturation. Therefore, some traditional theories suggest that denaturants should be avoided in SA-Biotin detection, as their use can lead to detection failure.

[0046] However, this invention has found that selecting appropriate denaturing agents can gently open up the spatial conformation, exposing the hidden biotinylated sites, without affecting the binding of other proteins due to steric hindrance or causing non-specific binding of non-biotinylated proteins. For example, this invention has found that by adding arginine and detergent, non-specific binding can be reduced, ensuring the accuracy of biotinylation rate detection. Within this window, the target protein can be effectively opened, reducing the influence of protein conformation on detection, while maintaining the high affinity binding activity of SA. This is an approach that introduces 'controlled denaturation' into 'functional detection,' which is fundamentally different from 'complete denaturation' in 'structural analysis.' Therefore, in preferred cases, the denaturing buffer is specifically composed of: PBS pH 6-9, 0.05M-0.5M Arginine, 0.01%-1% Tween 20, 4M-10M Urea; more specifically, it can be PBS pH 7, 0.05M Arginine, 0.01% Tween 20, 7M Urea; PBS pH 9, 0.1M Arginine, 0.1% Tween 20, 4M Urea, etc.

[0047] Of course, in some failed cases, this study found that not all denaturing agents or denaturing conditions can achieve the desired result. For example, when conventional thorough denaturation methods, such as SDS + boiling, were used, it was found that these denaturation methods did indeed completely destroy SA as traditional theory suggests, leading to detection failure.

[0048] Conditions for gender reassignment:

[0049] In some preferred methods, controlling the temperature at a low temperature, such as around 4°C, is more conducive to long-term incubation. This is because, when long-term incubation (such as overnight) is required, low temperature can minimize the activity of proteases and the chemical degradation of proteins themselves (such as deamidation and oxidation), thereby ensuring the accuracy and reproducibility of experimental results. In addition, some non-specific interactions are weakened at low temperatures.

[0050] However, according to our research, this denaturation process is actually not temperature-dependent. It is based on chemical denaturation, that is, driven by denaturing agents (urea, SDS, etc.), which breaks the non-covalent bonds of proteins and opens up the spatial structure. This process can be carried out effectively at room temperature or even higher temperatures, and is usually faster. In other words, when more efficient detection results are required, the temperature can be appropriately increased, while when a more stable reaction is required, low temperature conditions are preferred.

[0051] SI2. Using Nanodrop, detect the initial concentration of non-biotinylated protein (C1) and the concentration of biotinylated protein (BC1). Detect the same sample twice, and the concentration deviation should not exceed 10%. Otherwise, retesting is required.

[0052] In some experimental cases, with other conditions unchanged, step I was removed, i.e., the detection was performed using an invariant method, and the final biotin labeling rate was higher, even exceeding 100%.

[0053] II. Magnetic Bead Sealing (BSA)

[0054] SII1. Place the magnetic bead bottle on a vortex mixer for 20 seconds to resuspend the magnetic beads. Transfer 150 μL of magnetic beads (300 nm, 10 mg / ml) to a new centrifuge tube using a pipette. Incubate magnetically for 2 minutes, then remove the supernatant using a pipette and remove the centrifuge tube from the magnetic separator. Loading capacity: Biotinylated IgG ≥15 μg / mg magnetic beads

[0055] SII2. Add 300 μL of sterile water for injection to a centrifuge tube and mix the magnetic beads by pipetting. Separate magnetically and remove the supernatant.

[0056] SII3. Repeat SII2 twice, for a total of three washes.

[0057] SII4. Add 300 μL of BSA protein (10 mg / mL) diluted with sterile water for injection, and mix thoroughly by pipetting. Place the centrifuge tube on a constant temperature shaker and incubate at room temperature (100 rpm) for 60 min.

[0058] SII5. Remove the centrifuge tube, centrifuge for 5 seconds to separate magnetically, and discard the supernatant.

[0059] SII6. Add 600 μL of buffer to the centrifuge tube and thoroughly mix the magnetic beads by pipetting. Separate magnetically and remove the supernatant.

[0060] SII7. Repeat SII6 twice, for a total of three washes.

[0061] In this experimental example, BSA was used for blocking the magnetic beads. The purpose of this blocking experiment was to occupy all areas of the solid support (magnetic beads) except for the target binding site (streptavidin) with inert, non-reactive molecules. Any suitable protein or polymer that does not specifically bind to SA or biotin can achieve this goal. For example, in other cases, other inert proteins such as casein, skim milk powder, gelatin, and normal serum, as well as non-protein polymers such as polyethylene glycol and polyvinylpyrrolidone, have also been used to achieve the blocking objective.

[0062] III. Magnetic bead blocking (non-biotinylated protein)

[0063] SIII1. Use 75 μL of the non-biotinylated protein prepared in step I to resuspend the magnetic beads blocked by BSA in step II.

[0064] SIII2. Place the centrifuge tubes on a constant temperature shaker and incubate at room temperature (100 rpm) for 60 min.

[0065] SIII3. Remove the centrifuge tube, centrifuge for 5 seconds for magnetic separation, and transfer the supernatant to a new low-adsorption centrifuge tube. Vortex to mix, take 50 μL for the next experiment, and measure the concentration of the remaining portion (C2).

[0066] The difference between C2 and C1, which is the non-biotinylated protein that binds nonspecifically to BSA, needs to be less than 10% as a means of quality control during the process.

[0067] In some experimental cases, with other conditions unchanged and no sealing operation performed on the magnetic beads, the results were overestimated due to non-specific binding.

[0068] In other experimental cases, with other conditions unchanged, performing only the first type of blocking or only the second type of blocking resulted in higher results because either the binding of certain proteins to BSA could not be avoided, or the non-specific sites of the magnetic beads could not be avoided.

[0069] IV. Magnetic bead binding (biotinylated protein)

[0070] SIV1. Take 50 μL of the biotinylated protein prepared in step I and mix it with the supernatant transferred in step SIII3. Vortex to mix well, take 70 μL, resuspend the magnetic beads that were sealed in step III, and measure the concentration of the remaining part (M1).

[0071] Note: The M1 measured here is the actual value, and the deviation from the theoretical value should not exceed 0.3; otherwise, retesting is required. This requirement is to eliminate large errors in concentration detection. When testing unknown samples, the average value of multiple replicates can be taken.

[0072] SIV2. Place the centrifuge tubes on a constant temperature shaker and incubate at room temperature (100 rpm) for 60 min.

[0073] SIV3. Remove the centrifuge tube, centrifuge for 5 seconds for magnetic separation, and transfer the supernatant to a new low-adsorption centrifuge tube. Vortex to mix and measure the protein concentration (M2).

[0074] Unlike traditional magnetic beads used as 'capture tools' to detect target analytes, in this step they are used as 'measuring tools' to assess the quality of biotin labeling.

[0075] V. Data Processing

[0076] The biotinylated protein concentration difference before and after binding to the magnetic beads, divided by the initial biotinylated protein concentration, is the biotin labeling efficiency.

[0077] Calculation formula: Marking rate = (M1-M2) / (BC1 / 2)

[0078] Example 2. Anti-interference and stability test of the method in Example 1.

[0079] A. Non-biotinylated protein was added for interference. The biotinylated and non-biotinylated states of the same protein were pre-mixed at a specific mass ratio. After binding with SA magnetic beads, the results were detected as follows:

[0080]

[0081] The results show that the detection method is not affected by interference, and the actual detection labeling rate is basically consistent with the theoretical labeling rate, exhibiting a linear relationship.

[0082] B. Interference experiments with different protein samples, the results are as follows:

[0083]

[0084] The results show that the detection method is not affected by interference, and the actual detection labeling rate is basically consistent with the theoretical labeling rate, exhibiting a linear relationship.

[0085] C. Stability testing: This experiment used multiple samples and multiple tests. The test results are as follows:

[0086]

[0087] *This refers to different batches of the same protein.

[0088] The results show that for the same sample, the error is very small when detected at different time points. Furthermore, this method is applicable to a variety of different samples, and the detection results are stable with a CV% value of <5%.

[0089] In summary, the method of this embodiment shows that the detected values ​​are in high agreement with the theoretical values ​​and have a good linear relationship in the presence of interfering proteins; it is applicable to a variety of different protein samples and has strong universality; and the results of repeated detections are consistent.

[0090] Example 3. Comparative Experiment

[0091] The commonly used detection method on the market, the Pierce Biotin Quantitation Kit, utilizes the binding properties of HABA (4'-hydroxyazobenzene-2-carboxylic acid) with avidin to quantitatively determine the biotin content in a sample using spectrophotometry. In this comparative example, several commercially available kits were selected for the following tests:

[0092] Stability testing of commercially available reagent kits was conducted, and the results are as follows:

[0093]

[0094]

[0095]

[0096] Among them, commercially available product 1 is the commercially available P0371S Biotin Quantitative Assay Kit (Colorimetric Method), with the following specific components: P0371S-1: HABA / Streptavidin Premix, 2ml; P0371S-2: Reaction Buffer, 20ml; P0371S-3: Biotin Standard (500μM), 1ml; P0371S-4: Biotin-BSA (PositiveControl), 200μl;

[0097] Product 2 is the commercially available MAK171 colorimetric biotin assay kit, which consists of: Avidin, Catalog Number MAK171A, HABA Assay Buffer, Catalog Number MAK171B, and 100 µM D-Biotin Solution Catalog Number MAK171C.

[0098] The results showed that: for the same sample, test kits from different suppliers yielded different results; for the same sample, test kits from different batches yielded different results; and for the same sample, test kits from different testing times resulted in unstable test results and excessively high CV% values.

[0099] The reason for the above results may be that this method is essentially an indirect measurement and relies on biotin replacing HABA in the HABA / SA complex. The substitution reaction process may be the key to its instability. This invention provides a direct measurement based on strong interactions (Biotin-SA), fundamentally solving the stability problem. In other words, compared to the scheme in Example 1 of this experiment, the stability and interference resistance of existing kits are significantly inferior to this method. Therefore, the method of this embodiment is significantly superior to the HABA method.

[0100] The function and effect of this embodiment:

[0101] The reaction in this embodiment was carried out under denaturing conditions. Denaturing conditions ensured that the protein conformation was opened, allowing all biotinylate-labeled sites to be captured by streptavidin magnetic beads. This overcame steric hindrance, and the measured labeling efficiency is the "total labeling efficiency," resulting in more realistic and comparable results.

[0102] In this embodiment, the magnetic beads are specifically blocked. This is to reduce interference from non-specific binding on concentration determination. A two-step blocking method is employed, which significantly improves the signal-to-noise ratio and accuracy of the detection. Specifically, the first step is a general blocking, using an inert protein (such as BSA) to block possible non-specific binding sites on the surface of the magnetic beads. The second step is a specific competitive blocking, using the homologous non-biotinylated form of the analyte protein for secondary blocking. This effectively occupies protein regions that may weakly interact with the magnetic beads or buffer components, so that only the high-affinity SA-Biotin interaction is retained during subsequent incubation with biotinylated proteins, resulting in extremely low background signal.

[0103] In this embodiment, a direct calculation model based on the concentration difference (Δ value) is used: The present invention establishes a concise calculation formula: Labeling rate = (M1 - M2) / (BC1 / 2). Where M1 is the protein concentration in the supernatant before binding, M2 is the protein concentration in the supernatant after binding, and BC1 is the initial concentration of the biotinylated protein. This model has clear physical meaning, is directly calculated, and avoids errors caused by standard curve plotting.

[0104] While the foregoing has focused on embodiments, these are merely illustrative and do not limit the invention. Those skilled in the art will understand that various modifications and applications not illustrated above can be made without departing from the essential characteristics of these embodiments. For example, the constituent elements specifically shown in the embodiments can be implemented through modifications. Furthermore, various differences related to such modifications and applications should be interpreted as being included within the scope of the invention as defined in the appended claims.

Claims

1. A method for detecting the efficiency of protein biotin labeling, characterized in that: After denaturation, the protein was incubated with blocked magnetic beads. The labeling efficiency of biotin was calculated by measuring the concentration change of biotin before and after incubation with the magnetic beads. It includes the following steps: Step 1. According to the sample label concentration, dilute the non-biotinylated protein and biotinylated protein to the specified concentration with denaturing agent, and denature overnight at low temperature; Step 2. Detect the concentrations of non-biotinylated protein and biotinylated protein; Step 3. Use protein or polymer to seal the magnetic beads for the first time; Step 4. Perform a second blocking of the magnetic beads using non-biotinylated protein; Step 5. Bind the biotinylated protein to the magnetic beads and obtain the concentration values ​​of the biotinylated protein before and after binding to the magnetic beads; Step 6. Calculate biotin labeling efficiency.

2. The method for detecting protein biotin labeling efficiency as described in claim 1, characterized in that: The modification method is selected from at least one of the following methods AD: Method A. Add a chemical denaturant; Method B. Add ionic or nonionic detergents; Method C. Physical denaturation; Method D. Add a reducing agent.

3. The method for detecting protein biotin labeling efficiency as described in claim 1, characterized in that: The biotin labeling efficiency is calculated as the concentration difference of biotinylated protein before and after binding to the magnetic beads / initial biotinylated protein concentration / 2, which is the biotin labeling efficiency.

4. The method for detecting protein biotin labeling efficiency as described in claim 1, characterized in that: In step 4, the magnetic beads that have been blocked for the first time are resuspended in non-biotinylated protein and incubated at room temperature on a constant temperature shaker for 1-2 hours before magnetic separation.

5. The method for detecting protein biotin labeling efficiency as described in claim 1, characterized in that: In step 5, the biotinylated protein is resuspended in the magnetic beads that have been sealed for the second time, and then incubated on a constant temperature shaker at room temperature for 1-2 hours before magnetic separation.

6. A system for detecting the efficiency of protein biotin labeling, characterized in that: A means comprising performing the steps of the method as shown in any one of claims 1-5 in sequence.

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