Adiponectin antigen and antibody, preparation method of latex reagent thereof and adiponectin detection kit

A detection kit and adiponectin technology are applied in the adiponectin detection kit, the preparation of adiponectin antibody and adiponectin detection latex reagent, and the field of adiponectin antigen, which can solve the problems of poor test result accuracy and the like , to achieve wide applicability, accurate test results, and obvious effects.

Inactive Publication Date: 2019-10-25
HANGZHOU BOPU MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the lack of information about the preparation method of adiponectin antigen and adiponectin antibody in the prior art, and at the same time, the adiponectin polyclonal antibody in the adiponectin detection kit will interact with type VIII collagen, complement C1q and Ⅷ factor have cross-reactivity, which leads to the defect of poor accuracy of the final test results. It provides an effective preparation method for the preparation of adiponectin antigen and adiponectin antibody, and at the same time through the adiponectin antigen and adiponectin The antibody can be used to prepare adiponectin detection latex reagent and adiponectin detection kit

Method used

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  • Adiponectin antigen and antibody, preparation method of latex reagent thereof and adiponectin detection kit
  • Adiponectin antigen and antibody, preparation method of latex reagent thereof and adiponectin detection kit
  • Adiponectin antigen and antibody, preparation method of latex reagent thereof and adiponectin detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Preparation of adiponectin antigen:

[0050] 1. Adiponectin DNA sequence acquisition

[0051] 1.1 Obtain adiponectin DNA sequence from adiponectin positive tumor cells

[0052] Screen adiponectin-positive cell lines from tumor cells by immunoblotting, 1×10 6 For the tumor cells expressing adiponectin, the total RNA was isolated and purified with RNAeasy kit (purchased from Qiagen, Germany), and purified by Oligo dT 15 Primers (purchased from Shanghai Sangon Bioengineering Co., Ltd.) reverse-transcribed the total RNA into cDNA, and amplified the adiponectin DNA sequence (the nucleotide sequence is represented by SEQ ID NO.1) by polymerase chain reaction (PCR) method. shown, the amino acid sequence is shown in SEQ ID NO.2), DNA sequencing verification.

[0053] Its nucleotide sequence is as follows, denoted as SEQ ID NO.1:

[0054] 5'-atgctgttgc tgggagctgt tctactgcta ttagctctgc ccgggcatga ccaggaaacc 60acgactcaag ggcccggagt cctgcttccc ctgcccaagg gggcctgcac aggttggatg 1...

Embodiment 2

[0075] Preparation method of anti-adiponectin recombinant protein monoclonal IgG type antibody

[0076] 2.1 Animal immunity:

[0077] The adiponectin recombinant protein prepared above was mixed with an equal amount of complete Freund's adjuvant at a concentration of 100 mg adiponectin protein / time / mouse, and subcutaneously injected into immunized Balb / c female mice (5- 6 weeks old, body weight 18-20g), the interval between the first and the second time is 10-14 days, and thereafter each interval is 7 days, 5 consecutive times, the second-fifth time adiponectin protein and the same amount of incomplete Fu The adjuvant was emulsified and mixed well, and sterile adiponectin protein was injected into the tail vein / peritoneal cavity after 1 week to boost the immunization once, and the cells fused after 3 days.

[0078] 2.2 Preparation of hybridoma cells

[0079] The splenocytes of the immunized mice in step 2.1 were fused with SP2 / 0 myeloma cells at a volume ratio of 1:6 under t...

Embodiment 3

[0085] Preparation of latex reagent for adiponectin detection

[0086] 3.1 Preparation of preparation solution: solution A (25 mM MES buffer, pH=6.0), phosphate buffer (50 mM PB, pH=7.5), 2% BSA.

[0087] 3.2 Dilute 100nm latex microspheres with solution A to a concentration of 1%, and then combine with streptavidin at a ratio of 1:1 to obtain a microsphere solution containing streptavidin.

[0088] 3.3 The adiponectin monoclonal IgG antibody was diluted to a concentration of 3 mg / mL with phosphate buffer, and combined with biotin at a ratio of 1:1 to obtain a biotin-adiponectin monoclonal IgG antibody conjugate solution.

[0089] 3.4 Add the biotin-adiponectin monoclonal IgG antibody conjugate solution obtained in step (3.2) to the microsphere solution containing streptavidin obtained in step (3.1), wherein streptavidin and The ratio of biotin-adiponectin monoclonal IgG antibody conjugate is 1:1, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is dissolved in...

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Abstract

The invention relates to the technical field of biology, in particular to a preparation method of adiponectin antigen. The adiponectin DNA sequence is prepared by expressing positive tumor cells or achemical synthesis method, and then the adiponectin antigen is obtained by prokaryotic expression and eukaryotic cell expression. The adiponectin monoclonal antibody is prepared through animal immunization by taking the adiponectin monoclonal antigen as an immunogen and can be used for preparing an adiponectin detection latex reagent, and the prepared adiponectin detection latex reagent can be applied to an adiponectin detection kit. The adiponectin antigen overcomes the defects that the preparation method of adiponectin antigen and adiponectin antibody is unclear in the prior art, and simultaneously adiponectin polyclonal antibody in the adiponectin detection kit has cross reaction with type VIII collagen, complement C1q and type VIII factor, resulting in poor accuracy of the final test result. The method can effectively improve the test accuracy of the kit by introducing the adiponectin recombinant protein monoclonal type IgG antibody into the adiponectin detection kit.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method of an adiponectin antigen, an adiponectin antibody and a latex reagent for detecting adiponectin, and also relates to an adiponectin detection kit. Background technique [0002] Diabetes has become one of the three major chronic non-communicable diseases that threaten human health in the world. Although type II diabetes, which accounts for more than 95% of the total incidence, is widely distributed in the world, its prevalence also varies among different countries, regions and races. At present, the fact that the total number of diabetic patients in China ranks first in the world is indisputable, and the World Health Organization predicts that by 2025, the number of diabetic patients in the world will exceed 300 million, which will bring a very heavy burden to the entire society. [0003] Diabetes is also the culprit of many diseases. With the development of di...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K16/26C07K1/36C07K1/30C07K1/22C12N15/16C12N15/70C12N15/85G01N33/74G01N33/543
CPCC07K14/575C07K16/26C12N15/70C12N15/85G01N33/54313G01N33/74G01N2333/575G01N2800/042
Inventor 朱永良陈佳明程小龙丁洁
Owner HANGZHOU BOPU MEDICAL TECH
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