Preparation method of biotin-labeled German cockroach allergen I6 reagent

Abstract

The invention discloses a preparation method of a biotin-labeled German cockroach allergen I6 reagent, which comprises the following steps: 1) pretreating and extracting a German cockroach allergen I6 raw material to obtain an I6 protein solution; 2) based on the concentration of the I6 protein solution, mixing the I6 protein solution with a biotin labeling reagent for reaction to obtain a labeled I6 reagent; (3) carrying out purification treatment on the marked I6 reagent; before the step 2), carrying out surface modification on the I6 protein solution by using a low-temperature plasma treatment technology; and after the step 2), adding natural polysaccharide and the marked I6 reagent for compounding. According to the method, hydrophobic folds on the surface of protein are instantly opened through mild low-temperature plasma treatment, or some side chains are oxidized, more amino groups on lysine are exposed, the binding efficiency of biotin can be remarkably improved, and the quality guarantee period of an I6 reagent can be prolonged and the biological activity after labeling can be improved by using a natural polysaccharide compound.

Description

Technical Field

[0001] This invention relates to the field of I6 allergen reagent preparation technology, and in particular to a method for preparing a biotin-labeled German cockroach allergen I6 reagent. Background Technology

[0002] German cockroaches are a major allergen causing asthma and allergic rhinitis worldwide. With the development of component analysis diagnostic techniques, German cockroach allergen component I6, due to its high specificity, has become a key target for accurate diagnosis. In clinical testing, labeling the I6 protein using a biotin-streptavidin amplification system is a core method for improving the sensitivity of IgE antibody detection. However, preparing highly sensitive labeling reagents faces multiple challenges. First, the I6 protein reaction sites are mostly hidden in a hydrophobic core, resulting in low activation efficiency with traditional chemical methods. Second, conventional long-chain biotin easily creates steric hindrance on the protein surface, obscuring IgE binding sites. Furthermore, the hydrophobic groups introduced by labeling disrupt the protein hydration membrane, making the reagent highly susceptible to hydrophobic aggregation and precipitation in conventional systems.

[0003] In the prior art, patent CN103656635A proposes a method for preparing a German cockroach allergen formulation, including steps such as inactivation, washing, grinding, defatting, extraction, and addition of recombinant proteins. However, this technology only involves basic defatting and extraction, and does not solve the problem of low labeling rate caused by the concealment of the I6 protein reaction site, nor does it solve the problem of long-term stability after the introduction of hydrophobic groups into the label. Summary of the Invention

[0004] Purpose of the invention: In order to overcome the shortcomings of the existing technology, the present invention provides a method for preparing a biotin-labeled German cockroach allergen I6 reagent that can improve labeling efficiency and stability.

[0005] Technical solution: To achieve the above objectives, the present invention provides a method for preparing a biotin-labeled German cockroach allergen I6 reagent, comprising the following steps:

[0006] Step 1) Pre-treat and extract the German cockroach allergen I6 raw material to obtain I6 protein solution;

[0007] Step 2), based on the concentration of the I6 protein solution, the I6 protein solution is mixed with biotin-labeled reagent to obtain labeled I6 reagent;

[0008] Step 3) Purify the labeled I6 reagent;

[0009] Prior to step 2), the I6 protein solution is surface modified using low-temperature plasma treatment technology;

[0010] Following step 2), a natural polysaccharide is added and compounded with a labeled I6 reagent. The natural polysaccharide is hyaluronic acid or trehalose.

[0011] Furthermore, step 2) specifically includes the following steps:

[0012] Step 2.1) Determine the protein concentration of the I6 protein solution;

[0013] Step 2.2) Based on the protein concentration, concentrate or dilute the I6 protein solution to a concentration of 0.8-1.2 mg / ml, add 40 mM short-chain biotin at a molar ratio of 1:60-1:100, mix well, and label overnight on a shaker at a preset temperature.

[0014] Further, step 1) specifically includes:

[0015] Step 1.1) Degrease the I6 powder to obtain a degreased dry powder;

[0016] Step 1.2) Extract the defatted and dried powder to obtain I6 protein solution.

[0017] Further, step 1.1) specifically includes:

[0018] Step 1.1.1), weigh 1g of I6 powder and place it in a centrifuge tube;

[0019] Step 1.1.2): Add 30 mL of pre-cooled acetone (4°C) to the centrifuge tube, mix well, centrifuge and discard the supernatant; centrifugation parameters are: speed 5000 rpm, operation time 10 min, temperature 4°C.

[0020] Step 1.1.3), repeat the operation of step 1.1.2) until the supernatant is clear;

[0021] Step 1.1.4), take the precipitate from the centrifuge tube onto filter paper and place it in a fume hood for evaporation and drying.

[0022] Furthermore, step 1.2) specifically includes the following steps:

[0023] Step 1.2.1): Weigh an appropriate amount of dried powder, add 8-12mM PBS at a ratio of 1g / 15-25ml, mix well, and extract overnight on a shaker at 4℃.

[0024] Step 1.2.2), centrifuge and collect the supernatant, where the centrifugation parameters are: rotation speed 7000-9000 rpm, operation time 8-12 min, and temperature 2-6℃;

[0025] Steps 1, 2, and 3), pass through a 0.20-0.25 μm filter membrane.

[0026] Furthermore, step 2.1) specifically includes the following steps:

[0027] Step 2.1.1) Prepare BSA protein control solutions of 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, and 0.125 mg / ml, and serially dilute the extracted I6 protein solution;

[0028] Step 2.1.2) Add 200 μl of 1× Coomassie Brilliant Blue solution to every 20 μl of protein sample, mix well, and determine the protein concentration of the sample based on the color.

[0029] Furthermore, step 3) specifically includes:

[0030] Step 3.1) Take the labeled I6 reagent into a 4kD-6kD dialysis bag, place the dialysis bag in 3L of 10mM PBS solution and dialyze for about 24 hours, changing the solution 3 times during the period;

[0031] Step 3.2) Remove the I6 reagent from the dialysis bag, add an equal volume of glycerol, and store at -20°C.

[0032] Furthermore, the steps following step 3.1) and before step 3.2) also include:

[0033] The biotin labeling efficiency of the I6 allergen reagent after dialysis was verified, and the labeling rate was confirmed to be greater than 90% using the streptavidin-HRP detection system.

[0034] Beneficial effects: The method for preparing the biotin-labeled German cockroach allergen I6 reagent of the present invention has the following beneficial effects:

[0035] (1) The preparation method of the present invention, by using mild low-temperature plasma treatment to instantaneously open the hydrophobic folds on the protein surface or oxidize certain side chains to expose more amino groups on lysine residues, can significantly improve the biotin binding efficiency while avoiding breaking peptide bonds or destroying disulfide bonds, ensuring that the allergen retains its immune activity. The use of natural polysaccharide complexes can extend the shelf life of the I6 reagent, improve its bioactivity after labeling, make the allergen reagent more stable in complex application environments, and prevent it from losing its efficacy during storage and transportation.

[0036] (2) The preparation method of the present invention introduces short-chain biotin labeling, optimizes labeling efficiency and reduces protein degradation, thereby improving the sensitivity and specificity of IgE detection. The overall process is more standardized, reduces batch variation, is suitable for clinical diagnostic applications, and has an extraction repeatability error of less than 20%, significantly improving reagent reliability and diagnostic accuracy. Attached Figure Description

[0037] Figure 1A schematic flowchart of the preparation method of biotin-labeled German cockroach allergen I6 reagent;

[0038] Figure 2 This is a scatter plot of the clinical consistency analysis results. Detailed Implementation

[0039] The invention will now be further described with reference to the accompanying drawings.

[0040] like Figure 1 The method for preparing the biotin-labeled German cockroach allergen I6 reagent shown includes the following steps:

[0041] Step 1) Pre-treat and extract the German cockroach allergen I6 raw material to obtain I6 protein solution;

[0042] Step 2), based on the concentration of the I6 protein solution, the I6 protein solution is mixed with biotin-labeled reagent to obtain labeled I6 reagent;

[0043] Step 3) Purify the labeled I6 reagent;

[0044] Prior to step 2), the I6 protein solution is surface modified using low-temperature plasma treatment technology;

[0045] Following step 2), a natural polysaccharide is added and compounded with the labeled I6 reagent. The natural polysaccharide is hyaluronic acid or trehalose. This step improves the stability and efficiency of the labeling reaction and reduces the oxidation or degradation of the reagent. The concentration of the natural polysaccharide is 0.5%-2%.

[0046] Specifically, helium cold plasma jet treatment is employed, utilizing the generated gentle, reactive particle stream to impact the surface of the I6 protein. High-purity helium (greater than 99.99%) is preferred, as the helium-generated plasma is stable, low-temperature, and non-oxidizing. The treatment power is 10-30W, and the treatment time is 30-180 seconds. The jet nozzle is positioned 10-30mm from the liquid surface to avoid contact discharge. The I6 reagent is treated in PBS buffer. This working environment absorbs heat and excess free radicals, acting as a protective shield to further protect the protein structure. This physical impact and weak chemical modification temporarily disturb the hydration layer on the protein surface and slightly loosen the protein's tight folded structure, exposing lysine residues and their ε-amino groups, which were originally buried deep within the protein's hydrophobic core, to the surface. Since biotin-labeled reagents primarily target amino groups, more exposed sites directly improve coupling efficiency. Simultaneously, because the peptide bonds and disulfide bonds maintaining the protein backbone are not disrupted, the I6 protein rapidly recovers its native conformation after reconstitution, preserving intact IgE-binding epitopes.

[0047] The preparation method of this invention, through mild low-temperature plasma treatment, instantaneously opens the hydrophobic folds on the protein surface or oxidizes certain side chains, exposing more amino groups on lysine residues. This significantly improves biotin binding efficiency while avoiding breaking peptide bonds or disulfide bonds, ensuring the allergen retains its immune activity. The use of natural polysaccharide complexes extends the shelf life of the I6 reagent, enhances its bioactivity after labeling, and makes the allergen reagent more stable in complex application environments, less prone to losing efficacy during storage and transportation.

[0048] Preferably, step 2) specifically includes the following steps:

[0049] Step 2.1) Determine the protein concentration of the I6 protein solution;

[0050] Step 2.2) Based on the protein concentration, concentrate or dilute the I6 protein solution to a concentration of 0.8-1.2 mg / ml, add 40 mM short-chain biotin at a molar ratio of 1:60-1:100, mix well, and label overnight on a shaker at a preset temperature. In this embodiment, the concentration after concentration or dilution is 1 mg / ml, the molar ratio is 1:80, and labeling is performed overnight on a shaker at 4°C.

[0051] The preparation method of this invention introduces short-chain biotin labeling, optimizes labeling efficiency, reduces protein degradation, and improves the sensitivity and specificity of IgE detection. The overall process is more standardized, reducing batch variation and making it suitable for clinical diagnostic applications. Extraction repeatability error is less than 20%, significantly improving reagent reliability and diagnostic accuracy.

[0052] Preferably, step 1) specifically includes:

[0053] Step 1.1) Degrease the I6 powder to obtain a degreased dry powder;

[0054] Step 1.2) Extract the defatted and dried powder to obtain I6 protein solution.

[0055] Preferably, step 1.1) specifically includes:

[0056] Step 1.1.1), weigh 1g of I6 powder and place it in a centrifuge tube;

[0057] Step 1.1.2): Add 30 mL of pre-cooled acetone (4°C) to the centrifuge tube, mix well, centrifuge and discard the supernatant; centrifugation parameters are: speed 5000 rpm, operation time 10 min, temperature 4°C.

[0058] Step 1.1.3), repeat the operation of step 1.1.2) until the supernatant is clear;

[0059] Step 1.1.4), take the precipitate from the centrifuge tube onto filter paper and place it in a fume hood for evaporation and drying.

[0060] By following the steps above, we can ensure the complete removal of lipid interference, improve the purity and yield of allergen extraction, reduce batch variability, minimize the risk of protein degradation, and improve the efficiency and reagent stability of subsequent biotin labeling.

[0061] Preferably, step 1.2) specifically includes the following steps:

[0062] Step 1.2.1): Weigh an appropriate amount of dried powder, add 8-12mM PBS at a ratio of 1g / 15-25ml, mix well, and extract overnight on a shaker at 4℃.

[0063] Step 1.2.2), centrifuge and collect the supernatant, where the centrifugation parameters are: rotation speed 7000-9000 rpm, operation time 8-12 min, and temperature 2-6℃;

[0064] Steps 1, 2, and 3), pass through a 0.20-0.25 μm filter membrane.

[0065] Preferably, step 2.1) specifically includes the following steps:

[0066] Step 2.1.1) Prepare BSA protein control solutions of 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, and 0.125 mg / ml, and serially dilute the extracted I6 protein solution;

[0067] Step 2.1.2) Add 200 μl of 1× Coomassie Brilliant Blue solution to every 20 μl of protein sample, mix well, and determine the protein concentration of the sample based on the color.

[0068] In the above steps, by using an expanded range of BSA control solutions and optimizing the ratio of sample volume to Coomassie Brilliant Blue solution, more accurate concentration assessment was achieved, supporting subsequent labeling optimization, reducing measurement errors, and improving the reliability and diagnostic accuracy of the overall preparation process.

[0069] Preferably, step 3) specifically includes:

[0070] Step 3.1) Take the labeled I6 reagent into a 4kD-6kD dialysis bag, place the dialysis bag in 3L of 10mM PBS solution and dialyze for about 24 hours, changing the solution 3 times during the period;

[0071] Step 3.2) Remove the I6 reagent from the dialysis bag, add an equal volume of glycerol, and store at -20°C.

[0072] Preferably, the process after step 3.1 and before step 3.2 further includes:

[0073] The biotin labeling efficiency of the I6 allergen reagent after dialysis was verified, and the labeling rate was confirmed to be greater than 90% using the streptavidin-HRP detection system.

[0074] To verify the efficacy of the biotin-labeled I6 allergen reagent, its sensitivity and clinical consistency were validated:

[0075] (a) Sensitivity test

[0076] The sensitivity test results are shown in the table below:

[0077]

[0078] Sensitivity detection data show that, under low concentration sIgE gradient conditions (0, 0.1, 0.35 IU / mL), the detection rate and repeatability correlation coefficient (CV%) of the examples exhibit superior performance, and the values ​​at each concentration strictly meet the set standards.

[0079] The blank limit was initially assessed using the AVG+3SD method. The AVG+3SD of the example was 0.039 IU / mL, significantly lower than the measured value of 0.098 IU / mL corresponding to a concentration of 0.1 IU / mL. Therefore, the blank limit of the example was less than 0.1 IU / mL. In contrast, the measured blank limit of the comparative example, AVG+3SD = 0.116 IU / mL, was greater than its measured value of 0.094 IU / mL at 0.1 IU / mL. Therefore, the blank limit of the comparative example was higher than 0.1 IU / mL, and the sensitivity of the example was significantly higher than that of the comparative example.

[0080] When the sIgE concentration is 0 kUa / L, the corresponding value in the example is 14.4%, which is lower than the standard threshold of ≤15%, thus meeting the detection stability requirements at low concentrations.

[0081] When the sIgE concentration is 0.1 kUa / L, the corresponding value in the example is 9.7%, which is lower than the standard threshold of ≤10%, demonstrating good repeatability for low concentration detection;

[0082] When the sIgE concentration is 0.35 kUa / L, the corresponding value in the example is 6.5%, which is also lower than the standard threshold of ≤10%, further verifying its detection reliability in the low concentration range.

[0083] In contrast, the comparative examples, under the same sIgE concentration gradient, showed detection rates and repeatability correlation coefficients (CV%) of 20.3%, 17.6%, and 12.3%, respectively.

[0084] At 0 kUa / L, it was 5.9 percentage points higher than the example and exceeded the standard threshold of ≤15% by 5.3 percentage points;

[0085] At 0.1 kUa / L, it was 7.9 percentage points higher than the example, exceeding the standard threshold of ≤10% by 7.6 percentage points;

[0086] At 0.35 kUa / L, it is 5.8 percentage points higher than the example and exceeds the standard threshold of ≤10% by 2.3 percentage points.

[0087] The CV% values ​​of the comparative examples under various low concentration sIgE conditions were not only significantly higher than those of the examples, but also exceeded the corresponding standard thresholds. In contrast, the examples consistently met the detection requirements across the entire low concentration gradient range, demonstrating superior repeatability.

[0088] It is evident that the examples demonstrate significant advantages over the comparative examples in terms of measurement sensitivity and repeatability.

[0089] (II) Clinical Consistency Validation

[0090] The scatter plot of the analysis results of the clinical conformity trial is shown below. Figure 2 As shown.

[0091] The positive and negative composite rates of clinical data are shown in the table below:

[0092]

[0093] The test results show that the positive and negative consistency rate of the clinical data is over 90%. This data fully demonstrates that the two methods have a high degree of consistency in qualitative judgment (positive / negative). It means that the two methods are highly compatible in identifying the positive and negative status of the samples, reflecting that the two methods have good accuracy and reliability in qualitative detection and can stably reflect the actual positive and negative attributes of the samples.

[0094] Meanwhile, scatter plot analysis showed that the linear correlation coefficient (R²) of the detection values ​​of both methods reached above 0.990. This result indicates that, in the quantitative detection dimension, the detection results exhibit a very strong linear correlation; that is, when the detection value of one method changes, the detection value of the other method also shows a highly synchronous and regular change. This further confirms the high consistency of the two methods in quantitative results, indicating that they not only perform consistently in qualitative judgment but also have excellent agreement in the detection accuracy of specific values.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a biotin-labeled German cockroach allergen I6 reagent, comprising the following steps: Step 1) Pre-treat and extract the German cockroach allergen I6 raw material to obtain I6 protein solution; Step 2), based on the concentration of the I6 protein solution, the I6 protein solution is mixed with biotin-labeled reagent to obtain labeled I6 reagent; Step 3) Purify the labeled I6 reagent; Its features are: Prior to step 2), the I6 protein solution is surface modified using low-temperature plasma treatment technology; After step 2), natural polysaccharides are added and compounded with labeled I6 reagent.

2. The method for preparing the biotin-labeled German cockroach allergen I6 reagent according to claim 1, characterized in that, Step 2) specifically includes the following steps: Step 2.1) Determine the protein concentration of the I6 protein solution; Step 2.2) Based on the protein concentration, concentrate or dilute the I6 protein solution to a concentration of 0.8-1.2 mg / ml, add 40 mM short-chain biotin at a molar ratio of 1:60-1:100, mix well, and label overnight on a shaker at a preset temperature.

3. The method for preparing the biotin-labeled German cockroach allergen I6 reagent according to claim 1, characterized in that, Step 1) specifically includes: Step 1.1) Degrease the I6 powder to obtain a degreased dry powder; Step 1.2) Extract the defatted and dried powder to obtain I6 protein solution.

4. The method for preparing the biotin-labeled German cockroach allergen I6 reagent according to claim 3, characterized in that, Step 1.1) specifically includes: Step 1.1.1), weigh 1g of I6 powder and place it in a centrifuge tube; Step 1.1.2): Add 30 mL of pre-cooled acetone (4°C) to the centrifuge tube, mix well, centrifuge and discard the supernatant; Step 1.1.3), repeat the operation of step 1.1.2) until the supernatant is clear; Step 1.1.4), take the precipitate from the centrifuge tube onto filter paper and place it in a fume hood for evaporation and drying.

5. The method for preparing the biotin-labeled German cockroach allergen I6 reagent according to claim 3, characterized in that, Step 1.2) specifically includes the following steps: Step 1.2.1): Weigh an appropriate amount of dried powder, add 8-12mM PBS at a ratio of 1g / 15-25ml, mix well, and extract overnight on a shaker at 4℃. Step 1.2.2), centrifuge and collect the supernatant, where the centrifugation parameters are: rotation speed 7000-9000 rpm, operation time 8-12 min, and temperature 2-6℃; Steps 1, 2, and 3), pass through a 0.20-0.25 μm filter membrane.

6. The method for preparing the biotin-labeled German cockroach allergen I6 reagent according to claim 2, characterized in that, Step 2.1) specifically includes the following steps: Step 2.1.1) Prepare BSA protein control solutions of 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, and 0.125 mg / ml, and serially dilute the extracted I6 protein solution; Step 2.1.2) Add 200 μl of 1× Coomassie Brilliant Blue solution to every 20 μl of protein sample, mix well, and determine the protein concentration of the sample based on the color.

7. The method for preparing the biotin-labeled German cockroach allergen I6 reagent according to claim 1, characterized in that, Step 3) specifically includes: Step 3.1) Take the labeled I6 reagent into a 4kD-6kD dialysis bag, place the dialysis bag in 3L of 10mM PBS solution and dialyze for about 24 hours, changing the solution 3 times during the period; Step 3.2) Remove the I6 reagent from the dialysis bag, add an equal volume of glycerol, and store at -20°C.

8. The method for preparing the biotin-labeled German cockroach allergen I6 reagent according to claim 7, characterized in that, The steps following step 3.1) and before step 3.2) also include: The biotin labeling efficiency of the I6 allergen reagent after dialysis was verified, and the labeling rate was confirmed to be greater than 90% using the streptavidin-HRP detection system.

Images