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CN-121978324-A - Method for preparing biotin-labeled egg allergen F1 reagent

CN121978324ACN 121978324 ACN121978324 ACN 121978324ACN-121978324-A

Abstract

The invention discloses a method for preparing a biotin-marked egg allergen F1 reagent, which comprises the steps of a), preprocessing eggs to obtain clear egg white, b), mixing 0.2M pH 9.5 NaHCO 3 buffer solution with the same volume as the egg white, centrifuging to obtain supernatant, dialyzing the supernatant at 4 ℃ and then centrifuging to obtain egg white extract, C), carrying out buffer solution replacement and filtering operation on the egg white extract to obtain filtrate, d), adding a marking reagent into the filtrate to mark the filtrate to obtain a marked reagent, e), carrying out washing and centrifuging operation on the marked reagent for a plurality of times to obtain the F1 reagent, F), adding glycerol into the F1 reagent, sub-packaging and storing the F1 reagent, and b), dialyzing to obtain the egg white extract, wherein the method further comprises the steps of adjusting the pH of the egg white extract to about 4.0, heating at 60-65 ℃ to 15-30 min, rapidly cooling, centrifuging at 4 ℃ and at 12000 g to obtain precipitate, and collecting the supernatant to obtain further purified egg white extract.

Inventors

  • XU KE
  • ZHOU YUE
  • Song Juanyan
  • LIU JUN

Assignees

  • 东方伊诺(苏州)医疗科技有限公司

Dates

Publication Date
20260505
Application Date
20251208

Claims (10)

  1. 1. A method of preparing a biotin-labeled chicken egg allergen F1 reagent comprising the steps of: Step a), carrying out pretreatment on eggs to obtain clear egg white liquid; Step b), mixing 0.2M pH 9.5 NaHCO 3 buffer solutions with the same volume as egg white, centrifuging to obtain supernatant, dialyzing the supernatant at 4 ℃, and centrifuging to obtain egg white extract; step c), performing buffer replacement and filtration operation on the egg white extract to obtain filtrate; Step d), adding a marking reagent into the filtrate for marking to obtain a marked reagent; Step e) carrying out washing and centrifuging operation on the marked reagent for a plurality of times to obtain an F1 reagent; step F), adding the F1 reagent into glycerol, and then sub-packaging and storing; The method is characterized in that: The step b) of dialyzing the supernatant at 4 ℃ and then centrifuging to obtain an egg white extract, wherein the step b) further comprises the steps of adjusting the pH of the egg white extract to about 4.0, heating at 60-65 ℃ for 15-30 min, rapidly cooling, centrifuging at 4 ℃ and 12000 g for 10min, discarding the generated precipitate, and collecting the supernatant to obtain the further purified egg white extract.
  2. 2. A method for preparing a biotin-labeled chicken allergen F1 reagent as set forth in claim 1, wherein: In the step c), an ultrafiltration tube with the molecular weight cut-off of less than or equal to 10kDa is used for carrying out centrifugal concentration on the egg white extract, 10mM PBS buffer solution is added for a plurality of times for centrifugal replacement, and finally, the filtered solution is filtered by a 0.45 mu m filter membrane to obtain filtrate; The concentration of allergen protein in the filtrate was determined in step d) and labelled by reaction with 40 mM biotin overnight at 4℃in a molar ratio of 1:15-1:25.
  3. 3. The method for preparing a biotin-labeled chicken egg allergen F1 reagent according to claim 1, wherein the pretreatment of chicken eggs in step a) results in a clear egg white liquid comprising: Insoluble impurities such as egg yolk, eggshell fragments and viscous matters are removed by stirring egg white on ice and then passing through double-layer gauze.
  4. 4. The method for preparing a biotin-labeled chicken egg allergen F1 reagent as set forth in claim 1, wherein the buffer solution 0.2M pH 9.5 NaHCO 3 in step b) is prepared by the following steps: The amounts of the components were determined in equal proportions to the target amounts by adding 1.68g NaHCO 3 、12mL 0.5M NaHCO 3 and 50. Mu.l Proclin300 to 100mL of purified water in sequence to prepare a buffer.
  5. 5. The method for preparing a biotin-labeled chicken egg allergen F1 reagent as claimed in claim 1, wherein the low temperature centrifugation extraction method in step b) comprises: step b 1), centrifuging at a rotating speed of 3000r/min for 30min at a temperature of 4 ℃ to obtain a supernatant; step b 2), dialyzing in distilled water at 4 ℃ for 24 hours, and changing the solution for three times to obtain dialysate; And b 3) centrifuging the dialysate at a temperature of 4 ℃ for 10min with a parameter of 12000g, and taking supernatant to obtain the egg white extract.
  6. 6. The method for preparing a biotin-labeled chicken egg allergen F1 reagent as claimed in claim 2, wherein the 40 mM biotin in step d) is disposed as follows: Determining the dosage of each component according to the proportion of adding 1.4mg of the short-chain biotin of the aladine into each 100 mu l of DMSO or DMF and the like, and preparing a marking reagent; The calculation formula of the biotin consumption in the step d) is that B= (A multiplied by i)/(50000 multiplied by 40); wherein B is the amount of biotin in. Mu.l, A is the mass of the allergen protein calculated based on the concentration of the allergen protein, and i is the molar ratio of 15-25.
  7. 7. Method for the preparation of a biotin-labelled, egg allergen F1 reagent according to claim 1, characterized in that the labelled reagent is subjected to a plurality of washing centrifugation operations in step e), in particular comprising: Step e 1), adding the marked reagent into an ultrafiltration tube with the molecular weight cut-off of less than or equal to 3 kDa, and performing centrifugal operation; Step e 2), adding 10mM PBS and centrifuging four times; And e 3) reversely centrifuging to collect concentrated solution, adding 10mM PBS to blow the ultrafiltration tube membrane, reversely centrifuging, and combining to obtain the F1 reagent.
  8. 8. The method for preparing a biotin-labeled chicken egg allergen F1 reagent according to claim 1, wherein in the step b), after the pretreated egg white liquid is mixed with the 0.2M pH 9.5 NaHCO 3 buffer solution for extraction, absolute ethanol is added to the obtained extract to a volume fraction of 40-50%, and after standing at 4 ℃ for 30 minutes, centrifugation is performed at 3000 r/min for 30 min, the precipitate is discarded and the supernatant is retained as an egg white extract for the subsequent step.
  9. 9. The method for preparing a biotin-labeled chicken egg allergen F1 reagent as claimed in claim 1, wherein in the step d), after the completion of the biotin labeling reaction, a glycine solution of 0.1M and pH 7.4 is added to the reaction system to a final concentration of about 20 mM, and the reaction is left at room temperature for 30 to min to block unreacted active biotin ester, and then the centrifugal washing operation of the step e) is performed.
  10. 10. The method for preparing a biotin-labeled chicken egg allergen F1 reagent as claimed in claim 1, wherein in step F), trehalose is added as a stabilizer in an amount of 5-10% by mass to the F1 reagent, and the F1 reagent is freeze-dried under vacuum to obtain a freeze-dried F1 reagent for long-term storage.

Description

Method for preparing biotin-labeled egg allergen F1 reagent Technical Field The invention relates to the technical field of allergen detection reagent preparation, in particular to a method for preparing a biotin-marked egg allergen F1 reagent. Background Eggs are one of the common food allergens, and the incidence rate of egg allergy in people is high. Therefore, the obtained purified egg white allergen protein (such as the F1 protein mainly sensitized) has important significance in the food and medicine fields such as immunodiagnosis and the like. At present, various technical means such as organic solvent precipitation, aqueous two-phase extraction, electrophoretic separation, chromatographic separation, salting-out precipitation and the like are available for extracting and purifying egg white allergen proteins. However, these conventional methods often have problems of complicated steps, long time consumption, and low purity or yield. In the aspect of labeling preparation of allergen reagents, the common practice is to label the extracted allergen proteins with biotin and other molecules so as to be specifically combined with enzyme-labeled reporter molecules such as streptavidin in enzyme-linked immunosorbent assay and the like. In the prior art, patent CN108646024a discloses a food allergen ELISA detection reagent based on the antibody capturing principle, which uses a biotin-labeled crude extract of egg white allergen as a detection antigen in its design. By means of the streptavidin-biotin system, the method can detect a variety of food allergens, including eggs, representing the typical concept of allergen protein marker detection in the prior art. However, the above prior art still has significant limitations. First, since the crude allergen extract is directly used, the clarity and purity of the extract are insufficient, and the residual hetero protein may induce nonspecific reaction, affecting the detection specificity. Secondly, biotin randomly marks various components in the crude extract, so that marking uniformity is poor, part of target proteins are not marked enough, irrelevant components can be marked excessively, and effective activity and specificity of the reagent are reduced. And thirdly, the marked allergen reagent prepared from the crude extract has poor stability in the preservation process, active proteins are easy to degrade or lose activity, and the preparation has short validity period. Especially, the allergen content and activity of the natural raw material extracts of different batches are difficult to be consistent due to the source and preparation condition difference, so that the batch-to-batch difference is large. Disclosure of Invention The invention aims to overcome the defects in the prior art, and provides a method for preparing a biotin-marked egg allergen F1 reagent, which can ensure the clarity and purity of allergen extracts and ensure the purity and no impurities of target allergen components. Technical solution in order to achieve the above object, the method for preparing a biotin-labeled egg allergen F1 reagent of the present invention comprises the steps of: Step a), carrying out pretreatment on eggs to obtain clear egg white liquid; Step b), mixing 0.2M pH 9.5 NaHCO 3 buffer solutions with the same volume as egg white, centrifuging to obtain supernatant, dialyzing the supernatant at 4 ℃, and centrifuging to obtain egg white extract; step c), performing buffer replacement and filtration operation on the egg white extract to obtain filtrate; Step d), adding a marking reagent into the filtrate for marking to obtain a marked reagent; Step e) carrying out washing and centrifuging operation on the marked reagent for a plurality of times to obtain an F1 reagent; step F), adding the F1 reagent into glycerol, and then sub-packaging and storing; The step b) of dialyzing the supernatant at 4 ℃ and then centrifuging to obtain an egg white extract, wherein the step b) further comprises the steps of adjusting the pH of the egg white extract to about 4.0, heating at 60-65 ℃ for 15-30 min, rapidly cooling, centrifuging at 4 ℃ and 12000 g for 10min, discarding the generated precipitate, and collecting the supernatant to obtain the further purified egg white extract. Further, in the step c), an ultrafiltration tube with the molecular weight cut-off of less than or equal to 10kDa is used for carrying out centrifugal concentration on the egg white extract, 10mM PBS buffer solution is added for a plurality of times for centrifugal replacement, and finally, the filtered solution is filtered by a 0.45 mu m filter membrane to obtain filtrate; The concentration of allergen protein in the filtrate was determined in step d) and labelled by reaction with 40 mM biotin overnight at 4℃in a molar ratio of 1:15-1:25. Further, the pretreatment of the eggs in step a) results in a clear egg white liquid comprising: Insoluble impurities such as egg yolk, eggshell fragments and viscous matters