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Preparation method of NLS-RAR alpha monoclonal antibody, rapid detection card based on antibody and preparation method of rapid detection card

A monoclonal antibody and antibody technology, applied in the field of biomedicine, can solve the problems of high economic cost, long detection period, and high technical requirements of testing personnel, and achieve the effect of easy promotion.

Active Publication Date: 2022-04-12
AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These techniques are more objective and sensitive than morphology, but they also have many limitations: ① expensive equipment (requires flow cytometry, immunofluorescence microscope, etc.); ② long detection period (several days); ③ technical requirements High; ④High economic cost, heavy burden on patients; ⑤Bone marrow samples are required, peripheral blood cannot be detected, it is difficult to obtain materials, and it is traumatic to patients

Method used

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  • Preparation method of NLS-RAR alpha monoclonal antibody, rapid detection card based on antibody and preparation method of rapid detection card
  • Preparation method of NLS-RAR alpha monoclonal antibody, rapid detection card based on antibody and preparation method of rapid detection card
  • Preparation method of NLS-RAR alpha monoclonal antibody, rapid detection card based on antibody and preparation method of rapid detection card

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Embodiment 1

[0040] Preparation of NLS-RARα monoclonal antibody

[0041] NLS-RARα target protein sequence such as figure 1 As indicated, immunogens were selected and designed based on analysis of the structural properties of the NLS-RARα sequence, fragment immunogenicity, and water solubility.

[0042] The peptide immunogen sequence was determined, and the sequence is shown in Table 1. The COM-1a sequence VQSVPGAHPVPV-C is the 1-12 amino acid site of NLS-RARα, and the corresponding 1a polypeptide can specifically recognize the NLS-RARA specific polypeptide for the NE cleavage site of PML-RARα. The COM-1b sequence VPGAHPVPVYA-C is the 4-14 amino acid position of NLS-RARα. The corresponding 1b polypeptide has no specificity and can theoretically recognize both NLS-RARα and PML-RARα. It is mainly used for screening and control verification. After the sequence is determined, the immunogen is synthesized by artificial chemical methods. The synthesized peptide is analyzed by LC-MS for molecul...

Embodiment 2

[0054] Preparation of APL rapid detection card based on NLS-RARα monoclonal antibody

[0055] The NLS-RARα-specific monoclonal antibody was labeled with immunocolloidal gold, the precipitate was reconstituted with gold reconstitution solution, and spread on a 6mm×150mm gold pad to dry for later use. Coat the test line and quality control line on the NC membrane with NLS-RARα antibody stock solution and dilution solution, and dry it in low humidity for later use. Select the bottom plate, paste absorbent paper, NC membrane, gold pad, and sample chromatography pad in sequence from top to bottom, and assemble to obtain a rapid detection reaction plate for NLS-RARα. The schematic diagram of the principle of rapid detection technology is shown in Figure 7 , see the result judgment rules Figure 8 .

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Abstract

The invention provides a preparation method of an NLS-RAR alpha monoclonal antibody, a rapid detection card based on the antibody and a preparation method of the rapid detection card. The preparation method of the monoclonal antibody mainly comprises the following steps: determining an immunogen of targeted protein, carrying out experimental animal immunization, fusing spleen cells and myeloma cells of the immunized animal to obtain hybridoma cells, screening positive clones, and preparing a large number of monoclonal antibodies. And then preparing the APL rapid detection card based on the NLS-RAR alpha specific monoclonal antibody. The monoclonal antibody prepared by the invention is good in specificity; the APL rapid detection card based on the NLS-RAR alpha monoclonal antibody has the advantages of being minimally invasive, convenient, rapid and economical, can be used for peripheral blood detection, does not need special equipment, has the advantages which the existing diagnosis and treatment technology does not have, and is easier to popularize compared with the prior art.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a preparation method of NLS-RARα monoclonal antibody, a rapid detection card based on the antibody and a preparation method thereof. Background technique [0002] Acute promyelocytic leukemia (APL) belongs to the M3 subtype in the FAB classification, and is a special type of acute leukemia with specific chromosomal changes and fusion genes. Severe bleeding and disseminated intravascular coagulation (DIC) often occur in the early stage of APL, which has been considered as the most dangerous acute leukemia. Although the application of all-trans retinoic acid and arsenic targeted induction is effective, there are still 15% of APL patients Early death occurs due to late diagnosis. [0003] At present, the diagnosis of APL mainly relies on bone marrow morphology, chromosome detection, fluorescence in situ hybridization (FISH), immunophenotyping and fusion gene detection techniques. Bone m...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/20G01N33/543G01N33/577G01N33/68
CPCY02A50/30
Inventor 余莉华刘北忠钟梁徐婷万鹏但文冉
Owner AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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