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Preparation method of regenerated gene protein REG1 alpha monoclonal antibody

A monoclonal antibody and regenerative gene technology, applied in immunoglobulin, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve the problems of low sensitivity and specificity, achieve strong specificity, reduce Work load and time, and the effect of shortening the screening time of monoclonal antibodies

Pending Publication Date: 2020-11-10
甘肃智选生物科技有限公司
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At this stage, most of the antibodies for REG1α protein detection on the market are rabbit anti-polyclonal antibodies, which have relatively low sensitivity and specificity, while early clinical diagnostic samples are mainly serum and urine, which have a high sensitivity and specificity for detection. Higher requirements, therefore, a high titer monoclonal antibody against REG1α protein is the best way to improve the performance of the detection reagent

Method used

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Embodiment Construction

[0032] A preparation method of regenerative gene protein REG1α monoclonal antibody, comprising the following steps:

[0033] ⑴ Preparation of immunized mice:

[0034] Take recombinant REG1α protein and the same amount of complete Freund's adjuvant, mix well, inject into BCG-sensitized Balb / c mice subcutaneously in the groin and back of the neck respectively; each dose is 0.2mL, and the interval is 3 weeks, and the injection is repeated in the same way Twice, the immunized mice are obtained, and the tail tip blood of the immunized mice is taken two weeks after the last immunization for the determination of the antibody titer of the anti-REG1α protein in the mouse serum, and the antibody titer will be displayed Mice up to 1:20,000 were used as the source of immunized splenocytes; Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second and third times; The quantity is doubled, and the third time is double the prote...

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Abstract

The present invention relates to a preparation method of a regenerated gene protein REG1 alpha monoclonal antibody. The method comprises the following steps: (1) preparing immunized mice; (2) culturing myeloma cells for 3-4 days to obtain cultured mouse myeloma cells; (3) dissolving REG1 alpha protein, inoculating abdominal cavity of the immunized mice with the dissolved REG1 alpha protein for immunization, obtaining splenocytes, and dispersing the splenocytes to obtain a splenocyte precipitate; (4) centrifuging the cultured mouse myeloma cells to obtain a myeloma cell precipitate; (5) respectively suspending the splenocyte precipitate and the myeloma cell precipitate by using a DMEM high-glucose culture solution, conducting mixing, and centrifuging the mixture to obtain a precipitate; (6)centrifuging and suspending the precipitate to obtain fusion cells; (7) culturing and determining the fusion cells, and determining hybridoma cells; (8) screening and separating monoclonal antibodies; and (9) preparing a large amount of monoclonal antibodies, namely producing the monoclonal antibodies from a hybridoma cell line according to a conventional method. The preparation method is rapid and high in specificity.

Description

technical field [0001] The invention relates to a preparation method of a monoclonal antibody, in particular to a preparation method of a regenerative gene protein REG1α monoclonal antibody. Background technique [0002] Abnormal expression of the regeneration family gene (Reg) family is closely related to various human diseases such as inflammation and tumors, especially digestive system diseases. Clinically, abnormally increased Reg protein can often be detected in pathological specimens and serum of tumor patients, so Reg protein can be used as a clinical marker for predicting tumor progression and progression, especially for digestive system tumors, and can also be used as a diagnostic marker and therapeutic target genes. Reg1 is a member of the type I subclass of the Reg gene family. Fukui et al. found that the expression of Reg1 in gastric cancer tissues can reach 37.3%. Reg1 positive patients have higher proliferating cell nuclear antigen index than negative patients...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/577G01N33/569
CPCC07K16/18G01N33/577G01N33/56966C07K2317/33G01N2333/474
Inventor 连晓雯龚真莉赵晓虹霍锋李恩善王凯莉
Owner 甘肃智选生物科技有限公司
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