Human oophoroma tumor marker HE4 enzymoimmunoassay kit

An ovarian cancer and detection kit technology, applied in microorganisms, plant genetic improvement, botanical equipment and methods, etc., can solve problems such as limiting clinical application, and achieve the effects of simple operation, high cost performance and high stability

Inactive Publication Date: 2009-09-09
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

80% of patients with advanced ovarian cancer can detect the increase of CA125, but only 50-60% in the early stage, and CA125 wi...

Method used

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  • Human oophoroma tumor marker HE4 enzymoimmunoassay kit
  • Human oophoroma tumor marker HE4 enzymoimmunoassay kit
  • Human oophoroma tumor marker HE4 enzymoimmunoassay kit

Examples

Experimental program
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Effect test

Embodiment 1

[0014] Example 1: Construction, protein expression, purification and identification of HE4 gene

[0015] 1. Amplification of HE4 gene

[0016] According to the human HE4 mRNA sequence (NM_006103) provided in Genbank, 30 signal peptides at the N-terminus were removed, and 8 primers were designed and synthesized according to the preferred codons of Escherichia coli, respectively named P1-P8. The primers were synthesized by Dalian Bao Biological Company.

[0017] P1~P8 nucleotide sequence composition:

[0018] P1: tggttgtgctactttttgttctttacctaatgataaagaaggttcttgtc

[0019] P2: ctaattgaggaaaattaatattaacttgaggacaagaaccttctttatca

[0020] P3: tctgaatgtgctgataatttaaaatgttgttctgctggttgtgctacttt

[0021] P4: ttgagaatcaacttgacattgatcacgacataaacctaattgaggaaaat

[0022] P5: ctgatcaaaattgtactcaagaatgtgtttctgattctgaatgtgctgat

[0023] P6: caaccattacgacaacatttcatttgaccaggacattgagaatcaacttg

[0024] P7: cgggatccgaaaaaactggtgtttgtcctgaattacaagctgatcaaaattgta

[0025] P8: ccgctcgagtttatt...

Embodiment 2

[0052] Example 2: Preparation of rabbit anti-human HE4 polyclonal antibody

[0053] 1. Animal immunization

[0054] Take 500 μl (about 500 μg) of purified HE4 protein, fully emulsify it with an equal amount of Freund’s complete adjuvant, and immunize New Zealand big-eared white rabbits by subcutaneous multi-point immunization method on the back, and then immunize once every two weeks. The protein immunization dose is 300 μg, The adjuvant was Freund's incomplete adjuvant, and the whole process was immunized for 4 times, and blood was collected from the ear vein of rabbits seven days after the last immunization. After the antibody titer was determined by indirect ELISA, the carotid artery was bled, and the serum was collected and stored at -20°C.

[0055] 2. Purification and potency identification of rabbit anti-human HE4 polyclonal antibody

[0056] Rabbit serum was purified by Protein G affinity chromatography, and the serum was diluted with binding buffer (20mM sodium phosp...

Embodiment 3

[0059] Example 3: Preparation of mouse anti-human HE4 monoclonal antibody

[0060] 1. Animal immunization

[0061] Take 300 μl (about 300 μg) of purified HE4 protein and fully emulsify it with an equal amount of complete Freund’s adjuvant, inject BALB / c mice subcutaneously in multiple points on the back, and immunize 3 mice in total. One week later, each mouse was subcutaneously injected with 50 μg antigen dose and the same amount of Freund’s incomplete adjuvant fully emulsified, and then immunized once every two weeks. For intraperitoneal injection, from the second immunization, the mouse blood was collected from the tail vein on the seventh day after each immunization, and the serum titer was detected by indirect ELISA (the coated antigen was purified HE4 protein), and the titer reached 1:50000 After the above preparations for fusion, 3 days before fusion, a booster immunization was injected into the tail vein once, and the antigen dose was 50 μg.

[0062] 2. Establishment...

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Abstract

The invention discloses a human oophoroma tumor marker HE4 enzymoimmunoassay kit, a preparation method and an application thereof. A PCR overlapping method is adopted to fully manually synthesize an HE 4 gene by a synthesized HE4 gene prime; the gene is constructed into a Rho GEX-4T1 expression vector for protein expression; the expressed HE4 fusion protein is restricted by enzyme and purified to obtain an HE4 purified product which immunes animals to obtain a polyclonal antibody and a monoclonal antibody; a rabbit antihuman HE4 polyclonal antibody is used to wrap and close an ELISA plate; the rabbit polyclonal antibody is used as a capture antibody, rat antihuman HE4 monoclonal antibody labeled by horseradish peroxidase is used as a detection antibody so as to be assembled into a double-antibody sandwiched ELISA kit for detecting HE4. The kit has the advantages of high detection sensitivity and high specificity, is suitable for the early diagnosis of oophoroma and provides oophoroma patients for curative effect observation and prognosis judgment with an easy, convenient, rapid, economical and reliable detection method.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and disease detection and diagnosis reagent methods, in particular to HE4 gene synthesis, vector construction, protein expression and purification, HE4 used in ELISA kit and its preparation method. Background technique [0002] Ovarian cancer is one of the deadliest 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 to detect serum CA125. 80% of patients with advanced ovarian cancer can detect the increase of CA125, but only 50-60% in the early stage, and CA125 will also increase in some benign diseases, leading to some unnecessary operations, which greatly limits its use. Clinical application. Therefore, many sc...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/12C12N15/70C12P21/02G01N33/577G01N33/574C12R1/19
Inventor 李庆伟吴芬芳陈立勇王华颖王克威
Owner 大连美亿德生物科技有限公司
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