Time-resolved fluorescence (TRF) immunized detection kit of ovarian cancer tumor marker HE4

A time-resolved fluorescence, HE4 technology, applied in biological testing, anti-animal/human immunoglobulin, analytical materials, etc., can solve problems such as rising

Inactive Publication Date: 2011-01-19
大连美亿德生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CA125 is often not detected in the early stage of ovarian cancer, and can be detected in 80% of advanced patients, and CA125 will also increase in some benign diseases, leading to some unnecessary operations, and its clinical application has certain limitations

Method used

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  • Time-resolved fluorescence (TRF) immunized detection kit of ovarian cancer tumor marker HE4
  • Time-resolved fluorescence (TRF) immunized detection kit of ovarian cancer tumor marker HE4
  • Time-resolved fluorescence (TRF) immunized detection kit of ovarian cancer tumor marker HE4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Preparation of HE4 antigen

[0014] 1. Construction of pET-32a-HE4 recombinant vector

[0015] According to the human HE4 mRNA sequence (NM_006103) provided in GenBank, the N-terminal signal peptide was removed, a pair of primers were designed, and BamH I and Xho I restriction sites were introduced at the 5' ends of the two primers, respectively. The HE4 gene was extracted from the cancer cell line SKOV3, and agarose gel electrophoresis showed that the HE4 gene was successfully obtained, and the fragment length was consistent with the expectation ( figure 2 ).

[0016] The vector pET-32a(+) and the HE4 gene fragment purified by agarose gel were digested with BamH I and Xho I, and the digested product was purified and connected to obtain the recombinant plasmid pET-32a-HE4, the recombinant plasmid It was identified by double enzyme digestion ( figure 2 ), and the recombinant plasmid was sent to the company for sequencing. The sequencing results showed tha...

Embodiment 2

[0024] Example 2: Preparation of mouse anti-human HE4 monoclonal antibody

[0025] 1. Establishment of Hybridoma Cell Lines

[0026] Take 100μl (about 100μg) of purified HE4 protein and fully emulsify with the same amount of Freund's complete adjuvant, and inject BALB / c mice subcutaneously at multiple points on the back. The mice were injected subcutaneously at multiple points in the back, and then immunized once every two weeks. The immunization dose of each mouse was 50 μg without adjuvant. The immunization route was intraperitoneal injection. Starting from the second immunization, the tail vein was administered on the seventh day after each immunization The blood of mice was collected, and the serum titer reached 1:50,000 or more by indirect ELISA, and the mice were ready for fusion. Three days before fusion, a booster immunization was given by tail vein injection, and the antigen dose was 50 μg.

[0027] The mouse splenocytes were aseptically isolated during cell fusion, ...

Embodiment 3

[0034] Example 3: Establishment of Human Ovarian Cancer Tumor Marker HE4 Double Antibody Sandwich Time Resolved Fluorescence Immunoassay Method

[0035] 1. Detection method

[0036] In this example, the 6C2 monoclonal antibody obtained in Example 2 was used as the capture antibody, and Eu 3+ The labeled 5A3 monoclonal antibody was used as the detection antibody to establish a double-antibody sandwich time-resolved fluorescence immunoassay method for the detection of ovarian cancer tumor marker HE4.

[0037] 2. ELISA plate coating

[0038] Dilute the 6C2 monoclonal antibody to 10 μg / ml with coating solution (0.05mol / L carbonate buffer, pH 9.6), mix well, add 100 μl of the diluted monoclonal antibody solution to each well of the 96-well microtiter plate. The microtiter plate was sealed at 4°C overnight.

[0039] 3. ELISA plate blocking

[0040] PBS containing 20% ​​fetal bovine serum was used as blocking solution. The ELISA plate coated overnight was patted dry, 200 μl of b...

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Abstract

The invention relates to an analysis and detection technology of a human ovarian cancer tumor marker (human epididymis protein 4, HE4), in particular to a time-resolved fluorescence (TRF) immunized detection method and a kit of HE4, which is used for clinical auxiliary diagnosis, curative effect observation and prognosis judgment of the ovarian cancer. The invention comprises the following contents: constructing HE4 recombinant plasmids; expressing and purifying HE4 protein; immunizing BALB/c mice by the purified HE4 protein to prepare monoclonal antibodies; matching the obtained monoclonal antibodies to obtain two hybrid tumor cell strains (5A3 and 6C2) for secreting monoclonal antibodies of different epitopes of HE4 antigens, and marking the 5A3 monoclonal antibodies by the rare earth element Eu3+; and taking the unmarked 6C2 monoclonal antibodies as capture antibodies for coating a solid phase carrier, and taking the 5A3 monoclonal antibodies marked with the Eu3+ as detection antibodies to establish a double-antibody sandwich method for detecting HE4, thereby realizing the TRF immunized analysis of the invention.

Description

technical field [0001] The invention relates to an analysis and detection technology for human ovarian cancer tumor markers (epididymal secretory protein 4, HE4), in particular to a time-resolved fluorescence immunoassay method and detection kit for HE4, which are used for clinical auxiliary diagnosis of ovarian cancer, The invention relates to curative effect observation and prognosis judgment, belonging to the technical field of bioengineering and disease diagnosis methods. Background technique [0002] Ovarian cancer is one of the most lethal malignant tumors in women with insidious onset and poor prognosis. If ovarian cancer can be diagnosed at an early stage, the cure rate will be greatly improved, but ovarian cancer patients are often found at an advanced stage. At present, the most sensitive indicator for diagnosing ovarian cancer and monitoring its recurrence is the detection of serum CA125. However, CA125 is often undetectable in the early stage of ovarian cancer,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/574C07K16/18C07K14/47C12N15/12C12N15/70
Inventor 吴芬芳陈立勇王华颖
Owner 大连美亿德生物科技有限公司
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