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Double sandwich immunoassay test kit labeled by HE4 quantum dots and application thereof

A detection kit and immunoassay technology, applied in the field of immunodiagnosis, can solve the problems of low sensitivity and poor specificity, and achieve the effects of stable fluorescence, weak autofluorescence and long half-life

Inactive Publication Date: 2013-10-09
深圳市柏明胜医疗器械有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] One of the objectives of the present invention is to provide a human epididymis protein 4 (HE4) quantum dot-labeled double-sandwich immunoassay detection kit, which overcomes the low sensitivity and poor specificity of the existing HE4 enzyme immunoassay kits. technical flaw

Method used

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  • Double sandwich immunoassay test kit labeled by HE4 quantum dots and application thereof
  • Double sandwich immunoassay test kit labeled by HE4 quantum dots and application thereof
  • Double sandwich immunoassay test kit labeled by HE4 quantum dots and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: HE4 prokaryotic expression vector construction and protein expression, purification and identification

[0019] 1. Selection of HE4 immunogen

[0020] (1) HE4 gene immunogenic region

[0021] GAGAAGACTGGCGTGTGCCCCGAGCTCCAGGCTGACCAGAACTGCACGCAAGAGTGCGTCTCGGACAGCGAATGCGCCGACAACCTCAAGTGCTGCAGCGCGGGCTGTGCCACCTTCTGCTCTCTGCCCAATGATAAGGAGGGTTCCTGCCCCCAGGTGAACATTAACTTTCCCCAGCTCGGCCTCTGTCGGGACCAGTGCCAGGTGGACAGCCAGTGTCCTGGCCAGATGAAATGCTGCCGCAATGGCTGTGGGAAGGTGTCCTGTGTCACTCCCAATTTC

[0022] (2) Translation of the immunogenic region

[0023] EKTGVCPELQADQNCTQECVSDSECADNLKCCSAGCATFCSLPNDKEGSCPQVNINFPQLGLCRDQCQVDSQCPGQMK CCRNGCGKVSCVTP

[0024] 2. Construction of HE4 prokaryotic expression vector

[0025] (1) Restriction site and primer sequence

[0026] EcoRI Up primer: CCG gaattc gagaagactggcgtgtgcccc

[0027] XhoI Down primer: CCG ctcgag gaaattgggagtgacacagga

[0028] (2) Construction of HE4 prokaryotic expression vector

[0029] Mucinous ovarian cancer cells were...

Embodiment 2

[0037] Example 2: Preparation and identification of rabbit anti-human HE4 polyclonal antibody

[0038] 1. Animal immunity

[0039]Prepare two adult rabbits, dissolve 100μg antigen / rabbit into 1ml phosphate buffer solution for use. Add mycobacteria to 1ml of Freund's incomplete adjuvant to make a complete adjuvant, add 1ml of antigen solution, shake vigorously to make it fully emulsified, draw out the emulsion with a 3ml syringe, connect it with a 25G needle, and remove the air bubbles in the syringe . Rabbits were removed from the cage and placed on a flat surface, and subcutaneous injections were administered at 4 different sites, two on the back and two on the thigh. Brush the rabbit fur from the injection site and disinfect the exposed skin with ethanol. Pinch out the skin, insert the needle at an angle of 15 degrees relative to the skin, the depth of the needle is 1cm-2cm, be careful not to pierce the muscle, and inject about 500 μl of antigen solution in each of the 4 ...

Embodiment 3

[0062] Example 3: Preparation and identification of mouse anti-human HE4 monoclonal antibody

[0063] 1. Preparation of mouse anti-human HE4 monoclonal antibody

[0064] (1) Animal immunity

[0065] Immunization scheme: Fully emulsify the purified recombinant human HE4 antigen with an equal volume of Freund's complete adjuvant to make a water-in-oil antigen emulsion. SPF-grade pure-line BALB / c female mice aged 6-8 weeks, subcutaneously in multiple spots on the back injection. 100μg / rat, after two weeks, emulsify and immunize with the same amount of antigen plus the same volume of Freund's incomplete adjuvant. Booster immunizations were carried out at the end of the 2nd, 4th, and 6th weeks after the first immunization, and the same dose of the target protein was used each time. The titer of serum antibody was detected by indirect ELISA method. 3 days before cell fusion, mice were injected with 100 μg HE4 protein through tail vein or intraperitoneally to boost immunization. ...

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Abstract

The invention discloses a double sandwich immunoassay test kit labeled by HE4 quantum dots. The kit comprises HE4 protein standards, an ELISA plate coated with HE4 specific polyclonal antibodies, and CdTe quantum dots labeled monoclonal antibodies in specific binding with HE4 proteins. The invention further discloses an application of the kit in diagnosis of ovarian tumor. According to the invention, the test kit can directly determine test results through fluorescent ELISA, emitted fluorescence spectral peaks are narrow, autofluorescence is weak, and a sensitivity is high. The kit is high in fluorescence intensity and long in stable time, and can relatively effectively facilitate early detection and risk assessment of the ovarian tumor.

Description

technical field [0001] The invention relates to the technical field of immunodiagnosis, in particular to a human epididymis protein 4 (HE4) quantum dot double-sandwich immunoassay detection kit and its application. Background technique [0002] At present, two detection methods are generally used for ovarian cancer, but both have their limitations: 1) Transvaginal ultrasonography (TUV) cannot accurately detect benign and malignant tumors, and it requires experienced clinical technicians to detect Correct interpretation of the results; 2) The biomarker CA125 is now recognized as the "gold standard" for the diagnosis of ovarian cancer, but it also has its limitations. About 50% of patients with stage I ovarian cancer do not have elevated levels of CA125 , and certain benign ovarian diseases can also lead to elevated levels of CA125. The lower sensitivity and specificity of CA125 can lead to false negatives or false positives. Human epididymis protein 4 (HE4) belongs to the w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 陈平波徐洪斌夏曦方勇卢运萍申国辉杨绘
Owner 深圳市柏明胜医疗器械有限公司
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