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Hybridoma cell line scca1 and its secreted monoclonal antibody and application

A hybridoma cell line and monoclonal antibody technology, applied in the field of clinical diagnosis, can solve the problems of low sensitivity and low SCCA specificity, and achieve the effects of high sensitivity, fast detection speed and accurate detection results

Active Publication Date: 2019-01-25
RAYBIOTECH INC GUANGZHOU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, SCCA has the characteristics of low specificity and low sensitivity, and any lesion in the squamous epithelial cells of the whole body can make the serum SCCA test positive

Method used

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  • Hybridoma cell line scca1 and its secreted monoclonal antibody and application
  • Hybridoma cell line scca1 and its secreted monoclonal antibody and application
  • Hybridoma cell line scca1 and its secreted monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of hybridoma cell line SCCA1

[0025] Include the following steps:

[0026] 1. Preparation of antigen (SCCA1 recombinant protein)

[0027] Include the following steps:

[0028] 1), construction of pET28a-SCCA1 recombinant vector

[0029] According to the DNA sequence (NC_000018.10) of human SCCA1 (full length 1173bp) provided in Genbank, the sequence was artificially synthesized, the N-terminal signal peptide was removed, three pairs of primers were designed, and NdeI and XhoI were respectively introduced from the 5' ends of the two primers Restriction restriction site, four fragments of SCCA1 gene are amplified by conventional PCR method (vector pET-28a is template), respectively express four sections of sequence of SCCA1 protein: first section: the 93rd to the 159th amino acid (SEQ ID No: 1); second paragraph: amino acid 44 to 102 (SEQ ID No: 2); third paragraph: amino acid 52 to 112 (SEQ ID No: 3); fourth paragraph: full length , amino aci...

Embodiment 2

[0047] Example 2 Preparation and purification of mouse anti-SCCA ascites monoclonal antibody

[0048] Include the following steps:

[0049] a. Choose female healthy BALB / c mice aged 8-12 weeks, intraperitoneally inject pristane, 0.5mL / only; after 7-10 days, give each mouse intraperitoneal injection of 1*10 6 ~5*10 6 For the three monoclonal hybridoma cell lines SCCA1-1, SCCA1-2, and SCCA1-3 of Example 1, each monoclonal hybridoma cell was injected into two mice. Note that blowing off the cells from the culture dish or diluting the cells requires PBS or serum-free medium;

[0050] b. Centrifuge the ascitic fluid at 10,000r / min for 15 minutes to remove cell components and other sediments, fat and oil layers, and collect the middle layer;

[0051] c. Precipitation of saturated ammonium sulfate: absorb 5mL of the middle layer and transfer it into a small beaker, under stirring, add 5.0mL of PBS that has been filtered through a 0.22μm membrane dropwise; after mixing evenly, add ...

Embodiment 3

[0057] Example 3 Specific identification of the monoclonal antibody of Example 2

[0058] In this example, the SCCA1 recombinant antigen and CA125, CA153, CEA, AFP, HE4, GP73 proteins described in Example 1 were used as the coating antigen, and the monoclonal antibody prepared in Example 2 was used as the recognition antibody, SCCA was detected by indirect ELISA.

[0059] 1) Coating of the microtiter plate

[0060] Coating solution (Na 2 CO 3 1.5g, NaHCO 3 2.9g, Na 2 N 3 1.2g, add ddH 2 (0 to 1 L, adjust the pH to 9.6) Dilute the coated antigen to 1 μg / mL, mix well and add to a 96-well ELISA plate, 100 μL per well, seal the plate at 4°C overnight.

[0061] 2), the sealing of the enzyme plate

[0062] PBS containing 5% skim milk was used as blocking solution. First, pat dry the ELISA plate coated overnight, add 200 μL / well blocking solution, block at 37°C for 2 hours, wash the plate 6 times with a plate washer, pat dry the ELISA plate, and store it at 4°C for later ...

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Abstract

The invention discloses a hybridoma cell strain SCCA1 and its secreted monoclonal antibody and application thereof. The preservation number of the hybridoma cell strain SCCA1 is CCTCC No: C2016131, and the monoclonal antibody is secreted by the hybridoma cell strain SCCA1. Produced, the monoclonal antibody can be used in the preparation of reagents for detecting cervical cancer. The monoclonal antibody of the present invention has good specificity, and the in vitro diagnostic kit for detecting cervical cancer based on this has fast detection speed, less blood sample consumption, high sensitivity of detection results, and meets the different needs of clinic and practice. It provides a rapid, specific and sensitive detection method to greatly improve the monitoring and management of cervical cancer.

Description

technical field [0001] The invention belongs to the technical field of clinical diagnosis. Specifically, the invention relates to a hybridoma cell line SCCA1 and a preparation method thereof, a monoclonal antibody secreted by the hybridoma cell line SCCA1, and a monoclonal antibody in a test kit for detecting cervical cancer Applications. Background technique [0002] Cervical cancer is the third most common malignant tumor after breast cancer and colorectal cancer in women worldwide, and the second most common malignant tumor after breast cancer in developing countries, and is the most common cancer of the female reproductive tract. Malignant tumor. [0003] Cervical cancer refers to malignant tumors that occur at the junction of squamous epithelial cells of the cervicovaginal or transition zone and columnar epithelial cells of the endocervical canal. After decades of extensive gynecological screening, the prevalence and mortality of cervical cancer have been reduced by n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/30G01N33/574G01N33/577C12R1/91
CPCC07K16/30C12N1/00C12R2001/91G01N33/574G01N33/577
Inventor 黄若磐罗树红方建民张玉明胡洋
Owner RAYBIOTECH INC GUANGZHOU
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