Method for preparing biotin-labeled egg allergen F1 reagent

Abstract

The invention discloses a method for preparing a biotin-labeled egg allergen F1 reagent, which comprises the following steps of: a) pretreating an egg to obtain clear egg white liquid; b) mixing 0.2 M NaHCO3 buffer solutions with the same volume as the egg white and the pH value of 9.5, centrifuging to take supernate, dialyzing the supernate at the temperature of 4 DEG C, and then centrifuging to obtain an egg white extracting solution; c) performing buffer solution replacement and filtration operation on the egg white extracting solution to obtain filtrate; d) adding a labeling reagent into the filtrate for labeling to obtain a labeled reagent; e) washing and centrifuging the marked reagent for multiple times to obtain an F1 reagent; f) adding the reagent F1 into glycerol, and then sub-packaging and storing; wherein after the egg white extracting solution is obtained through dialysis in the step b), the pH value of the egg white extracting solution is adjusted to be about 4.0, then the egg white extracting solution is heated for 15-30 min at the temperature of 60-65 DEG C, the egg white extracting solution is rapidly cooled and then centrifuged for 10 min at the temperature of 4 DEG C and the weight of 12000 g, generated precipitates are discarded, supernatant is collected, and the egg white extracting solution which is further purified is obtained.

Description

Technical Field

[0001] This invention relates to the field of allergen detection reagent preparation technology, and in particular to a method for preparing a biotin-labeled egg allergen F1 reagent. Background Technology

[0002] Eggs are a common food allergen, and the incidence of egg allergy is relatively high in the population. Therefore, obtaining purified egg white allergen proteins (such as the major sensitizing F1 protein) is of great significance in food and pharmaceutical fields such as immunodiagnostics. Currently, there are various techniques for the extraction and purification of egg white allergen proteins, such as organic solvent precipitation, aqueous two-phase extraction, electrophoretic separation, chromatographic separation, and salting-out precipitation. However, these traditional methods often suffer from complex procedures, long processing times, and low purity or yield.

[0003] In the preparation of labeled allergen reagents, a common practice is to label the extracted allergen proteins with molecules such as biotin, so that they can specifically bind to enzyme-labeled reporter molecules such as streptavidin in enzyme-linked immunosorbent assays (ELISA). In the prior art, patent CN108646024A discloses a food allergen ELISA detection reagent based on the antibody capture principle, which uses a biotin-labeled crude extract of egg white allergens as the detection antigen. Through the streptavidin-biotin system, this method can detect multiple food allergens, including eggs, representing a typical approach to allergen protein labeling detection in the prior art.

[0004] However, the aforementioned existing technologies still have significant limitations. First, because crude allergen extracts are used directly, the clarity and purity of the extracts are insufficient, and residual proteins may trigger non-specific reactions, affecting the detection specificity. Second, the random labeling of various components in the crude extract with biotin results in poor labeling uniformity; some target proteins are underlabeled, while irrelevant components may be overlabeled, reducing the effective activity and specificity of the reagent. Third, labeled allergen reagents prepared from crude extracts exhibit poor stability during storage; active proteins are easily degraded or lose their activity, resulting in a short shelf life. In particular, due to differences in source and preparation conditions, the allergen content and activity of different batches of natural raw material extracts are difficult to maintain consistently, leading to large batch-to-batch variations. Summary of the Invention

[0005] Purpose of the invention: In order to overcome the shortcomings of the existing technology, the present invention provides a method for preparing biotin-labeled egg allergen F1 reagent that can improve the clarity and purity of allergen extracts and ensure that the target allergen components are pure and free of impurities.

[0006] Technical solution: To achieve the above objective, the present invention provides a method for preparing a biotin-labeled egg allergen F1 reagent, comprising the following steps:

[0007] Step a) Pre-treat the eggs to obtain a clear egg white liquid;

[0008] Step b) Mix egg white with an equal volume of 0.2M pH 9.5 NaHCO3 buffer, centrifuge to obtain the supernatant, dialyze the supernatant at 4°C and then centrifuge again to obtain egg white extract;

[0009] Step c) Perform buffer replacement and filtration on the egg white extract to obtain the filtrate;

[0010] Step d) Add a labeling reagent to the filtrate to label it, and obtain the labeled reagent;

[0011] Step e) Perform multiple washing and centrifugation operations on the labeled reagent to obtain reagent F1;

[0012] Step f) After adding the F1 reagent to glycerol, dispense and store it.

[0013] After dialysis of the supernatant at 4°C and centrifugation to obtain egg white extract in step b), the process further includes: adjusting the pH of the egg white extract to approximately 4.0, then heating it at 60–65°C for 15–30 min, and rapidly cooling it before centrifuging it at 4°C and 12000 g for 10 min. The resulting precipitate is discarded, and the supernatant is collected to obtain a further purified egg white extract.

[0014] Further, in step c), the egg white extract is concentrated by centrifugation using an ultrafiltration tube with a molecular weight cutoff of ≤10kDa, and 10mM PBS buffer is added multiple times for centrifugation replacement. Finally, the filtered solution is filtered through a 0.45μm filter membrane to obtain the filtrate.

[0015] In step d), the concentration of allergen proteins in the filtrate is determined, and the filtrate is reacted with 40 mM biotin at a molar ratio of 1:15-1:25 overnight at 4°C for labeling.

[0016] Further, the pretreatment of the eggs described in step a) to obtain a clear egg white liquid includes:

[0017] To remove insoluble impurities such as egg yolk, eggshell fragments, and viscous substances, the method for removing viscous substances is to stir the egg whites on ice and then pass them through two layers of gauze.

[0018] Furthermore, the preparation method for the 0.2M pH 9.5 NaHCO3 buffer solution in step b) is as follows:

[0019] Prepare the buffer solution by adding 1.68g NaHCO3, 12mL 0.5M NaHCO3, and 50μl Proclin 300 to every 100mL of pure water, proportionally determining the amount of each component according to the target dosage. In actual preparation, prepare a larger amount of buffer solution than the target dosage, and then mix the target dosage of the buffer solution with the egg white solution.

[0020] Furthermore, the low-temperature centrifugation extraction method in step b) specifically includes:

[0021] Step b1), centrifuge at 3000 r / min for 30 min at 4℃ and collect the supernatant;

[0022] Step b2): Dialyze the solution in distilled water at 4°C for 24 hours, changing the solution three times during the process, to obtain the dialysate.

[0023] Step b3) The dialysate is centrifuged at 4°C with a parameter of 12000g for 10 minutes, and the supernatant is collected to obtain the egg white extract.

[0024] Furthermore, the 40 mM biotin in step d) is prepared as follows:

[0025] The labeling reagent was prepared by adding 1.4 mg of Aladdin short-chain biotin to every 100 μl DMSO or DMF, and determining the amount of each component proportionally according to the target dosage.

[0026] The formula for calculating the amount of biotin used in step d) is: B = (A × i) / (50000 × 40);

[0027] Where B is the amount of biotin used, in μl; A is the mass of allergen protein calculated based on the allergen protein concentration, in mg; and i is the molar ratio, with a value of 15-25.

[0028] Further, step e) involves performing multiple washing and centrifugation operations on the labeled reagent, specifically including:

[0029] Step e1): Add the labeled reagent to an ultrafiltration tube with a molecular weight cutoff of ≤3 kDa and centrifuge.

[0030] Step e2), add 10mM PBS and centrifuge four times;

[0031] Step e3): Invert the tube and centrifuge to collect the concentrated solution. Add 10 mM PBS and agitate the ultrafiltration membrane. Invert the tube and centrifuge again to combine the solutions to obtain reagent F1. After obtaining reagent F1, the reagent concentration can be tested using a micro spectrophotometer.

[0032] Further, in step b), after mixing the pretreated egg white solution with the 0.2M pH 9.5 NaHCO3 buffer solution for extraction, anhydrous ethanol is added to the resulting extract to a volume fraction of 40–50%, and the mixture is allowed to stand at 4°C for 30 minutes. Then, it is centrifuged at 3000 r / min for 30 min, the precipitate is discarded and the supernatant is retained as the egg white extract for subsequent steps.

[0033] Further, in step d), after the biotin labeling reaction is completed, a 0.1 M glycine solution at pH 7.4 is added to the reaction system to a final concentration of about 20 mM, and the system is allowed to react at room temperature for 30 min to block the unreacted active biotin ester, and then the centrifugation and washing operation of step e) is performed.

[0034] Further, in step f), 5–10% by mass of trehalose is added to the F1 reagent as a stabilizer, and the F1 reagent is freeze-dried under vacuum conditions to obtain freeze-dried F1 reagent for long-term storage.

[0035] Beneficial effects: The method for preparing biotin-labeled egg allergen F1 reagent of the present invention has the following beneficial effects:

[0036] (1) This invention utilizes the unique heat and acid resistance properties of egg allergen F1 protein to perform acid-heat treatment on the protein extract obtained after dialysis. That is, by combining alkaline extraction at pH 9.5 with heat treatment at 60–65℃ under pH 4.0 conditions, heat-sensitive impurities such as ovalbumin are specifically denatured, precipitated, and removed. This realizes the reverse elimination of impurities by utilizing the specific biochemical properties of F1 protein, and realizes the purification logic of first alkali and then calculation, first dissolution and then precipitation, thereby obtaining a highly clear purified extract.

[0037] (2) Based on the highly clear purified extract, biotin labeling was performed using an optimized low molar ratio (1:15–1:25), which effectively avoided non-specific adsorption caused by over-labeling, significantly reduced background signal and improved detection sensitivity. The addition of glycine blocking and trehalose lyophilization technology further ensured the long-term stability and batch-to-batch consistency of the reagent. Attached Figure Description

[0038] Figure 1 A schematic flowchart of a method for preparing biotin-labeled egg allergen F1 reagent;

[0039] Figure 2 This is a scatter plot showing the results of clinical consistency analysis. Detailed Implementation

[0040] The invention will now be further described with reference to the accompanying drawings.

[0041] like Figure 1The method for preparing biotin-labeled egg allergen F1 reagent shown includes the following steps:

[0042] Step a) Pre-treat the eggs to obtain a clear egg white liquid;

[0043] Step b) Mix egg white with an equal volume of 0.2M pH 9.5 NaHCO3 buffer, centrifuge to obtain the supernatant, dialyze the supernatant at 4°C and then centrifuge again to obtain egg white extract; specifically, after mixing egg white with alkaline buffer, stir at room temperature for 1 hour and then let stand in the refrigerator overnight, and then perform low-temperature centrifugation extraction.

[0044] Step c) The egg white extract is subjected to buffer replacement and filtration to obtain a filtrate. Specifically, the egg white extract is concentrated by centrifugation using an ultrafiltration tube with a molecular weight cutoff of ≤10kDa, and 10mM PBS buffer is added multiple times for centrifugation replacement. Finally, the filtered solution is filtered through a 0.45μm filter membrane to obtain a filtrate. In this embodiment, the specific steps are as follows: First, the egg white extract is concentrated by centrifugation using an ultrafiltration tube with a molecular weight cutoff of ≤10kDa at 12000g for 10min. Then, pure water is added, the mixture is inverted and mixed, and centrifuged twice at 12000g for 10min. Finally, the liquid in the tube is diluted with an appropriate amount of 10mM PBS and filtered through a 0.45μm microporous membrane.

[0045] Step d) Add a labeling reagent to the filtrate to label it, and obtain the labeled reagent; specifically, the concentration of allergen protein in the filtrate is determined, and it is labeled by reacting with 40 mM biotin at 4°C overnight at a molar ratio of 1:15-1:25, preferably a molar ratio of 1:20.

[0046] Step e) Perform multiple washing and centrifugation operations on the labeled reagent to obtain reagent F1;

[0047] Step f) After adding the F1 reagent to glycerol, aliquot and store at -20°C;

[0048] The main innovation of this invention lies in the following: After obtaining the egg white extract through dialysis in step b), the pH of the egg white extract is adjusted to approximately 4.0, then heated at 60–65°C for 15–30 min, rapidly cooled, and centrifuged at 12000 g for 10 min at 4°C. The resulting precipitate is discarded, and the supernatant is collected to obtain a further purified egg white extract. By appropriately acidifying the extract and subjecting it to medium-temperature heat treatment, the heat resistance of major allergen proteins (such as the main component in egg white allergen F1) is utilized to denature and precipitate residual impurity proteins (such as other heat-sensitive egg white components or microaggregates formed after dialysis) and remove them, resulting in a high-purity, high-clarity allergen extract. This further reduces background interference and improves the efficiency and product stability of the biotinylated reaction.

[0049] Furthermore, the use of ultrafiltration tubes enables simultaneous protein concentration and precise buffer replacement, significantly improving the purity of the target protein. Optimized conditions, including a specific molar ratio and overnight labeling with 40 mM biotin at 4°C, ensure efficient and stable labeling reactions, reducing byproducts. Subsequent removal of free biotin further guarantees the homogeneity of the labeled products. This method significantly improves the sensitivity, specificity, and batch-to-batch consistency of allergen-labeled products, meeting the requirements for high-precision immunoassay.

[0050] Preferably, the pretreatment of eggs in step a) to obtain a clear egg white liquid includes:

[0051] To remove insoluble impurities such as egg yolk, eggshell fragments, and viscous substances, the method for removing viscous substances is to stir the egg whites on ice and then pass them through two layers of gauze.

[0052] Preferably, the 0.2M pH 9.5 NaHCO3 buffer solution in step b) is prepared as follows:

[0053] Prepare the buffer solution by adding 1.68g NaHCO3, 12mL 0.5M NaHCO3, and 50μl Proclin 300 to every 100mL of pure water, proportionally determining the amount of each component according to the target dosage. In actual preparation, prepare a larger volume of buffer solution than the target dosage, and then mix the target volume of buffer solution with the egg white solution. For example, if the target dosage is 190mL, take 200mL of pure water and add 3.36g NaHCO3, 24mL 0.5M NaHCO3, and 100μl Proclin 300 sequentially. The total volume of the resulting buffer solution will be greater than 200mL; use only 190mL of this solution.

[0054] In the above buffer preparation method, precise pH adjustment can be achieved by first adding NaHCO3 solid and then fine-tuning with concentrated solution. Adjusting the pH to 9.5 avoids the isoelectric point of egg white allergens, overcomes the viscous matrix of egg white, improves extraction efficiency, inhibits protease activity, reduces target protein degradation, maintains conformational integrity, and ensures efficient biotin labeling. Adding Proclin 300 provides long-lasting inhibition of microbial growth, ensures the chemical stability of the extraction buffer, prevents target protein degradation or modification, and ultimately maintains batch-to-batch consistency and detection reliability of the allergen reagent.

[0055] Preferably, the low-temperature centrifugation extraction method in step b) specifically includes:

[0056] Step b1) Centrifuge at 3000 r / min for 30 min at 4℃ and collect the supernatant; the initial centrifugation speed in this step is relatively low, which can gently remove large particulate impurities and avoid excessive shear damage.

[0057] Step b2) Dialyze the solution in distilled water at 4°C for 24 hours, changing the solution three times during the process, to obtain the dialysate; this step can completely remove salt ions and small molecule interfering substances.

[0058] Step b3): The dialysate is centrifuged at 12000g for 10 minutes at 4°C, and the supernatant is collected to obtain the egg white extract. This high-speed centrifugation effectively removes colloidal precipitates and microaggregates formed after dialysis.

[0059] The above steps, through the synergistic effect of three-step centrifugation and dialysis, significantly improve the efficiency and purity of protein extraction, solve the problem of stubborn turbidity in traditional processes, and finally obtain egg white extract with high clarity and low background interference, providing an ideal raw material basis for subsequent labeling reactions.

[0060] Preferably, the 40 mM biotin in step d) is prepared as follows:

[0061] The labeling reagent was prepared by adding 1.4 mg of Aladdin short-chain biotin to every 100 μl DMSO or DMF, and determining the amount of each component proportionally according to the target dosage.

[0062] The formula for calculating the amount of biotin used in step d) is: B = (A × i) / (50000 × 40);

[0063] Where B is the amount of biotin used, in μl; A is the mass of allergen protein calculated based on the allergen protein concentration, in mg; and i is the molar ratio, with a value of 15-25.

[0064] For example, in this embodiment, i=20, and taking A=0.5mg as an example, B=5μl is calculated.

[0065] Preferably, step e) involves multiple washing and centrifugation operations on the labeled reagent, specifically including:

[0066] Step e1): Add the labeled reagent to an ultrafiltration tube with a molecular weight cutoff of ≤3 kDa and centrifuge it. The centrifugation parameters are 12000g and 10min.

[0067] Step e2), add 10mM PBS, centrifuge four times, with centrifugation parameters of 12000g, 10min;

[0068] Step e3): Invert the tube and centrifuge to collect the concentrated solution. Add 10 mM PBS and agitate the ultrafiltration membrane. Invert the tube and centrifuge again to combine the solutions to obtain reagent F1. After obtaining reagent F1, the reagent concentration can be tested using a micro spectrophotometer.

[0069] Preferably, in step b), after mixing the pretreated egg white solution with the 0.2M pH 9.5 NaHCO3 buffer for extraction, anhydrous ethanol is added to the resulting extract to a volume fraction of 40–50%. The mixture is then incubated at 4°C for 30 minutes, centrifuged at 3000 r / min for 30 min, the precipitate is discarded, and the supernatant is retained as the egg white extract for subsequent steps. By introducing an ethanol precipitation step after alkaline extraction, most impurities other than the target allergen protein are selectively precipitated, significantly reducing the probability of hybrid proteins being co-labeled subsequently. This yields a clear extract rich in F1 allergen, reduces non-specific background, and improves the purity and specificity of the labeled product.

[0070] Preferably, in step d), after the biotin labeling reaction is completed, a 0.1 M glycine solution at pH 7.4 is added to the reaction system to a final concentration of approximately 20 mM, and the reaction is allowed to proceed at room temperature for 30 min to block unreacted active biotin esters. Then, the centrifugation and washing operation of step e is performed. By introducing a glycine quenching step after the labeling reaction, the remaining active biotin reagent is neutralized, preventing it from continuing to react nonspecifically with the target protein or causing side reactions during subsequent storage. This ensures the structural integrity and uniformity of the labeled product, reduces nonspecific background signals, and improves the stability and reproducibility of the reagent.

[0071] Preferably, in step f), 5–10% by mass of trehalose is added to the F1 reagent as a stabilizer, and the F1 reagent is freeze-dried under vacuum conditions to obtain freeze-dried F1 reagent for long-term storage. By adding trehalose and performing vacuum freeze-drying, liquid biotin-labeled allergens are prepared into stable freeze-dried formulations, significantly improving the stability of the reagent during transportation and storage. Trehalose and other protective agents effectively protect the conformation of the allergen protein, and its immunomodulatory activity remains good after freeze-drying and reconstitution, thus ensuring batch-to-batch consistency and detection reliability of the allergen F1 reagent.

[0072] Experimental results

[0073] To evaluate the technical effectiveness of this method, the sensitivity and clinical consistency of the F1 reagent prepared in this embodiment and the F1 reagent prepared by the conventional method were verified.

[0074] The table below shows the results of the sensitivity verification.

[0075]

[0076] Sensitivity evaluation results showed that, at low sIgE gradients (0, 0.1, 0.35 kUa / L), the examples demonstrated superior performance in both detection rate and repeatability (CV%), with all indicators meeting and exceeding the established standards.

[0077] First, the blank limit was estimated using the AVG+3SD method: the blank limit of the example was 0.039 IU / mL, significantly lower than the measured value of 0.103 IU / mL for the 0.1 IU / mL concentration sample, therefore its blank limit was clearly less than 0.1 IU / mL; the blank limit of the comparative example was 0.105 IU / mL, actually higher than the measured value of 0.104 IU / mL for its 0.1 IU / mL concentration sample, meaning its blank limit exceeded 0.1 IU / mL. Therefore, it is evident that the sensitivity of the example is significantly better than that of the comparative example.

[0078] Secondly, regarding repeatability (CV%), the example maintained high stability at all three low concentration points: at 0 kUa / L, CV% = 14.4%, lower than the standard requirement of ≤15%; at 0.1 kUa / L, CV% = 7.9%, far below ≤10%; and at 0.35 kUa / L, CV% = 5.4%, also meeting the ≤10% requirement. In contrast, the comparative example's CV% under the same concentration conditions were 24.6%, 17.9%, and 13.5%, respectively: at 0 kUa / L, it was 10.2 percentage points higher than the example and exceeded the standard limit by 9.6 percentage points; at 0.1 kUa / L, it was 10.0 percentage points higher than the example and exceeded the standard limit by 7.9 percentage points; and at 0.35 kUa / L, it was 8.1 percentage points higher than the example and exceeded the standard threshold by 3.5 percentage points.

[0079] Overall, the comparative example not only had a higher CV% across all low concentration ranges than the example, but also exceeded the corresponding limits in all cases. In contrast, the example remained stable and compliant with standards across the entire low concentration range, demonstrating its significant advantages in sensitivity and repeatability.

[0080] Figure 2 The results of the scatter analysis show the clinical consistency.

[0081] The table below shows the positive and negative concordance rates of the protein (F1) allergen-specific IgE antibody detection kit.

[0082]

[0083] According to the test results, the positive and negative concordance rates of the clinical samples were all above 90%, indicating that the two methods have high consistency in the qualitative interpretation of negative / positive results. In other words, they are basically synchronized in identifying the positive and negative status of the samples, and can accurately and stably reflect the true positive and negative attributes of the samples, demonstrating good reliability and accuracy at the qualitative detection level.

[0084] also, Figure 2 Scatter plot analysis showed that the linear correlation coefficients (R²) of the detection results for both methods were greater than 0.990. This high correlation indicates that, at the quantitative detection level, the numerical trends of the two methods are highly consistent: when the detection value of one method increases or decreases, the result of the other method shows a corresponding and regular change. This result further proves that the two methods not only have good consistency in qualitative judgment but also exhibit excellent matching and consistency at the specific quantitative numerical level.

[0085] 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 egg allergen F1 reagent, comprising the following steps: Step a) Pre-treat the eggs to obtain a clear egg white liquid; Step b) Mix egg white with an equal volume of 0.2M pH 9.5 NaHCO3 buffer, centrifuge to obtain the supernatant, dialyze the supernatant at 4°C and then centrifuge again to obtain egg white extract; Step c) Perform buffer replacement and filtration on the egg white extract to obtain the filtrate; Step d) Add a labeling reagent to the filtrate to label it, and obtain the labeled reagent; Step e) Perform multiple washing and centrifugation operations on the labeled reagent to obtain reagent F1; Step f) After adding the F1 reagent to glycerol, dispense and store it. Its features are: After dialysis of the supernatant at 4°C and centrifugation to obtain egg white extract in step b), the process further includes: adjusting the pH of the egg white extract to approximately 4.0, then heating it at 60–65°C for 15–30 min, and rapidly cooling it before centrifuging it at 4°C and 12000 g for 10 min. The resulting precipitate is discarded, and the supernatant is collected to obtain a further purified egg white extract.

2. The method for preparing biotin-labeled egg allergen F1 reagent according to claim 1, characterized in that: In step c), the egg white extract was concentrated by centrifugation using an ultrafiltration tube with a molecular weight cutoff of ≤10kDa, and 10mM PBS buffer was added multiple times for centrifugation replacement. Finally, the filtered solution was filtered through a 0.45μm filter membrane to obtain the filtrate. In step d), the concentration of allergen proteins in the filtrate is determined, and the filtrate is reacted with 40 mM biotin at a molar ratio of 1:15-1:25 overnight at 4°C for labeling.

3. The method for preparing biotin-labeled egg allergen F1 reagent according to claim 1, characterized in that, The pretreatment of eggs described in step a) to obtain a clear egg white liquid includes: To remove insoluble impurities such as egg yolk, eggshell fragments, and viscous substances, the method for removing viscous substances is to stir the egg whites on ice and then pass them through two layers of gauze.

4. The method for preparing biotin-labeled egg allergen F1 reagent according to claim 1, characterized in that, The preparation method for the 0.2M pH 9.5 NaHCO3 buffer solution in step b) is as follows: Prepare the buffer solution by adding 1.68g NaHCO3, 12mL 0.5M NaHCO3, and 50μl Proclin300 to every 100mL of pure water in that order, and determining the amount of each component proportionally according to the target usage.

5. The method for preparing biotin-labeled egg allergen F1 reagent according to claim 1, characterized in that, The low-temperature centrifugation extraction method in step b) specifically includes: Step b1), centrifuge at 3000 r / min for 30 min at 4℃ and collect the supernatant; Step b2): Dialyze the solution in distilled water at 4°C for 24 hours, changing the solution three times during the process, to obtain the dialysate. Step b3) The dialysate is centrifuged at 4°C with a parameter of 12000g for 10 minutes, and the supernatant is collected to obtain the egg white extract.

6. The method for preparing biotin-labeled egg allergen F1 reagent according to claim 2, characterized in that, The preparation method for 40 mM biotin in step d) is as follows: The labeling reagent was prepared by adding 1.4 mg of Aladdin short-chain biotin to every 100 μl DMSO or DMF, and determining the amount of each component proportionally according to the target dosage. The formula for calculating the amount of biotin in step d) is: B = (A × i) / (50000 × 40); Where B is the amount of biotin used, in μl; A is the mass of allergen protein calculated based on the allergen protein concentration, in mg; and i is the molar ratio, with a value of 15-25.

7. The method for preparing biotin-labeled egg allergen F1 reagent according to claim 1, characterized in that, Step e) involves performing multiple washing and centrifugation operations on the labeled reagent, specifically including: Step e1): Add the labeled reagent to an ultrafiltration tube with a molecular weight cutoff of ≤3 kDa and centrifuge. Step e2), add 10mM PBS and centrifuge four times; Step e3): Invert the tube and centrifuge to collect the concentrate. Add 10 mM PBS and blow the ultrafiltration membrane. Invert the tube and centrifuge again to combine the solutions to obtain reagent F1.

8. The method for preparing biotin-labeled egg allergen F1 reagent according to claim 1, characterized in that, In step b), the pretreated egg white solution is mixed with the 0.2M pH 9.5 NaHCO3 buffer solution for extraction. Anhydrous ethanol is added to the resulting extract to a volume fraction of 40–50%. After standing at 4°C for 30 minutes, the mixture is centrifuged at 3000 r / min for 30 minutes. The precipitate is discarded and the supernatant is retained as the egg white extract for subsequent steps.

9. The method for preparing biotin-labeled egg allergen F1 reagent according to claim 1, characterized in that, In step d), after the biotin labeling reaction is completed, a 0.1 M glycine solution at pH 7.4 is added to the reaction system to a final concentration of about 20 mM. The system is then allowed to react at room temperature for 30 min to block the unreacted active biotin esters. Then, the centrifugation and washing operation of step e) is performed.

10. The method for preparing biotin-labeled egg allergen F1 reagent according to claim 1, characterized in that, In step f), 5–10% by mass of trehalose is added to the F1 reagent as a stabilizer, and the F1 reagent is freeze-dried under vacuum conditions to obtain freeze-dried F1 reagent for long-term storage.

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