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Kit for detecting endoplasmic reticulum membrane protein complex subunit 10 in human serum

An endoplasmic reticulum membrane and complex technology is applied in the field of kits for detecting endoplasmic reticulum membrane protein complex subunit 10 in human serum, and can solve the problem that quantitative detection is not very accurate.

Active Publication Date: 2021-05-11
上海伦泽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, except Wang Xuanchun’s team, there is no literature report that EMC10 can be detected in human serum. The research group previously established an ELISA method using a rabbit anti-human EMC10 polyclonal antibody, and detected the presence of EMC10 in human serum (qualitatively). In this method, the EMC10 polyclonal antibody is used as both a coating antibody and a detection antibody, so the quantitative detection of EMC10 in serum is not very accurate, and the detected content of EMC10 in human serum is 1-10ng / mL

Method used

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  • Kit for detecting endoplasmic reticulum membrane protein complex subunit 10 in human serum
  • Kit for detecting endoplasmic reticulum membrane protein complex subunit 10 in human serum
  • Kit for detecting endoplasmic reticulum membrane protein complex subunit 10 in human serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, the preparation of EMC10 monoclonal antibody

[0078] 1. Construction of EMC10 eukaryotic expression recombinant plasmid

[0079] (1) The full-length EMC10 cDNA was cloned by PCR, and NheI and XhoI restriction sites were introduced at both ends of the fragment.

[0080] Using the DNA fragment shown in SEQ ID No.2 as a template, and using EMC10-F and EMC10-R as primers, a product of about 700 bp was amplified by PCR, and the PCR product was recovered by gel and purified.

[0081] EMC10-F: 5'-CTA GCTAGC AAGCGGCTGCCGGGCCGGGACTG-3';

[0082] EMC10-R: 5'-CCG CTCGAG GGCCTCCTGTGGCGGTGGCG-3'.

[0083] (2) The product was recovered by NheI and XhoI double-enzyme digestion gel, and ligated into the eukaryotic expression vector pRAG2a digested with the same enzyme, T4 DNA ligase was ligated to construct a recombinant plasmid, and transformed into TOP10 competent cells.

[0084] (3) PCR identification of positive clones: identification of correct clones was ver...

Embodiment 2

[0160] Example 2, EMC10 different antibody pairing ELISA double antibody sandwich method to detect the content of EMC10 in human serum

[0161] First, the purpose

[0162] The EMC10 ELISA double antibody sandwich method was used to detect the content of EMC10 in human serum.

[0163] 2. Detection principle

[0164] Coat the microwell plate with the purified antibody to make a solid phase carrier, add the specimen or standard, biotinylated anti-EMC10 antibody, and HRP-labeled avidin to the microwells coated with the anti-EMC10 antibody in sequence, and thoroughly After washing, the color was developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The depth of the color is positively correlated with the EMC10 in the sample. Measure the absorbance (OD value) at 450 / 630nm dual wavelength with a microplate reader, and calculate the sample concentration.

[0165] 3. Experimental equipmen...

Embodiment 3

[0261] Embodiment 3, the preparation of EMC10 quantitative detection kit (chemiluminescence method)

[0262] 1. Comparison of 1F12 monoclonal antibody and 6B9 monoclonal antibody as capture antibodies in chemiluminescent immunoassay (CLIA)

[0263] Based on the results obtained in Example 2, in the ELISA method, "1F12 monoclonal antibody as capture antibody, 6B9-Biotin monoclonal antibody as detection antibody" is the optimal combination with the best effect. The inventor first used the method of chemiluminescence immunoassay (CLIA) "1F12 monoclonal antibody as capture antibody, 6B9 monoclonal antibody as detection antibody" to quantitatively detect the content of EMC10 in a series of human serum samples to be tested (see Table 9). For specific operations, refer to the relevant steps in step 2 below.

[0264] However, it can be seen from the test results that there are still some human sera that cannot detect EMC10 by this method (Table 9). For this reason, the inventors imp...

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Abstract

The invention discloses a kit for detecting the subunit 10 of the endoplasmic reticulum membrane protein complex in human serum. The invention provides a test kit for detecting EMC10 content in human serum, including a capture antibody (anti-EMC10 monoclonal antibody 6B9, secreted by mouse hybridoma cell line 6B9 CGMCC No.15789) and a detection antibody (anti-EMC10 Monoclonal antibody 1F12, secreted by mouse hybridoma cell line 1F12 CGMCC No.15788). The present invention uses the monoclonal antibody secreted by the mouse hybridoma cell line 6B9 as the capture antibody, and the monoclonal antibody secreted by the mouse hybridoma cell line 1F12 as the detection antibody to prepare a kit for detecting the EMC10 content in human serum. EMC10 was successfully detected in the serum of the Chinese population and the Caucasian population, and the detection line was very low, and this method has been very stable.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a kit for detecting subunit 10 of endoplasmic reticulum membrane protein complex in human serum. Background technique [0002] Endoplasmic reticulum membrane protein complex subunit 10 (ER membrane protein complex subunit 10, EMC10) was initially cloned by Wang Xuanchun et al. The acid sequence and its encoded amino acid sequence were delivered to the GenBank database (accession number: AY194293) [1]. Wang Xuanchun's team reported the cloning and preliminary functional study of the INM02 gene for the first time in the world, and the article has been published in the "Journal of Endocrinology" [2]. Since EMC10 has no significant homology to any known protein or protein domain, EMC10 is considered a novel protein. Wang Xuanchun's team has been engaged in the research of EMC10. After nearly 10 years of hard work, it has revealed a variety of biological functions of EMC10. Using the ELI...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/535G01N33/558G01N33/577
CPCG01N33/535G01N33/558G01N33/577
Inventor 杨志红赵伟
Owner 上海伦泽生物科技有限公司
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