HE4 (Human Epididymis Protein) monoclonal and polyclonal antibody preparation and development of corresponding diagnostic reagent kit

A diagnostic kit, HE4 technology, applied in the direction of anti-animal/human immunoglobulin, measuring devices, instruments, etc., can solve the problems of ineffective early diagnosis of ovarian cancer, reduce specificity, etc., to improve sensitivity and Effects of specificity, simplicity of materials, and easy availability of materials

Inactive Publication Date: 2011-01-19
TIANJIN GOALGEN BIOTECH
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

Although the above combined method may improve the detection sensitivity, it is at the cost of reduc

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Recombinant expression, purification and identification of HE4

[0020] The HE4 gene was cloned from ovarian cancer tissue by RT-PCR, inserted into the vector pPICZaA, positive clones were picked, recombinant plasmid DNA was extracted, and enzyme digestion analysis was performed with XhoI, XbaI, BamHI and HindIII. Enzyme-digested fragments with the expected size; after sequencing confirmed the correct gene, it was transformed into Pichia pastoris GS115 and KM71, and yeast engineering bacteria were screened, and the secreted expression product of recombinant yeast was analyzed by SDS-PAGE, and it was found to be specifically expressed at about 20-25kD Band, western blotting analysis proves that the expression product can specifically bind to HE4 monoclonal antibody.

Embodiment 2

[0022] Preparation, purification and identification of anti-HE4 antibody

[0023] 1) Preparation, purification and identification of monoclonal antibody against HE4

[0024]BALB / c mice were immunized with purified HE4 by intraperitoneal injection, once every three weeks, and immunized four times in total; then the spleen cells of the immunized mice were taken out, and PEG-4000 was used to fuse the spleen cells with the bone tumor of the mice Cell line P3-X63-Ag8.653, using HAT selection medium to select hybridoma cells on a 96-well cell culture plate, and using ELISA to identify cells that produce anti-HE4 antibodies; save the screened cells that specifically bind to HE4 Monoclonal antibody cell line; use cell fermentation equipment to culture the preserved cell line, collect the cell culture supernatant, and then use Protein A-Sepharose affinity chromatography column to further purify the anti-HE4 monoclonal antibody from the cell culture supernatant, SDS -PAGE and western b...

Embodiment 3

[0028] Development of ELISA Diagnostic Kit

[0029] 1) ELISA plate selection: select a polystyrene plate with good adsorption performance, low blank value, high transparency at the bottom of the well, and similar performance between each plate and between each well of the same plate;

[0030] 2) Coating: Use carbonate buffer solution with pH 9.6 as the coating diluent, add the coating solution to the wells of the ELISA plate, place it in the refrigerator at 4-8°C overnight, and keep it at 37°C for 2 hours. The same coating effect. Generally, the coating concentration of protein is 100ng / ml-20ug / ml;

[0031] 3) Blocking: use 1% gelatin solution as the blocking solution;

[0032] 4) Enzyme substrate solution: 60ug / ml TMB, 0.045% H 2 o 2 In 0.1mol / L pH6.0 phosphate buffer;

[0033] 5) Stop solution: 2mol / L H 2 SO 4 solution;

[0034] 6) Washing solution: 0.01 mol / L pH 7.4 phosphate-NaCl buffer solution containing 0.05% Tween-20.

[0035] 7) Operation steps

[0036] To d...

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Abstract

The invention relates to a preparation method of a diagnostic reagent kit for early diagnosis and recurrence monitoring, and the like of ovarian cancer. The preparation method comprises the following steps of: the recombination expression of a marker HE4 (Human Epididymis Protein) in eukaryotic cells, monoclonal and polyclonal antibody preparation by HE4 obtained by the recombination expression in the eukaryotic cells, and the diagnostic reagent kit (a double antibody sandwich method and an enzyme-linked immunization method) development by the prepared monoclonal and polyclonal antibodies. The invention can ensure that HE4 obtained by the recombination expression in the eukaryotic cells by utilizing a gene engineering method has the same glycosylation degree with natural HE4; the space conformation thereof more approaches to a natural antigen; the prepared monoclonal and polyclonal antibody is used as a coating antibody, and the other monoclonal antibody is used as an enzyme mark antibody to develop the reagent kit; and the sensitivity and the specificity are higher.

Description

Technical field: [0001] The present invention relates to the technical field of preparation of HE4 antibody (including monoclonal antibody and polyantibody), recombinant expression of HE4, early diagnosis of ovarian cancer and recurrence monitoring of ovarian cancer by using HE4 antibody. Background of the invention: [0002] Ovarian cancer is a common malignant tumor in gynecology, including several different histopathological types, and the treatment method depends on the specific tumor type. Among them, epithelial ovarian cancer accounts for the majority of malignant tumors, and also includes others such as germ cell tumors, carcinosarcoma (malignant Müllerian mixed tumor of the ovary) and ovarian stromal tumors. Less than 40% of patients with epithelial ovarian cancer can be cured, and most of them are in the advanced stage when they are first diagnosed. [0003] According to WHO's recent worldwide statistics, the morbidity and mortality of ovarian cancer are only lower...

Claims

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Application Information

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IPC IPC(8): G01N33/577C07K16/18C07K16/06
Inventor 郝振明周贞德
Owner TIANJIN GOALGEN BIOTECH
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