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Tandem immuno-assay for cancer

A technology of immunoassay and assay method, applied in immunoglobulin, anti-enzyme immunoglobulin, microbial assay/inspection, etc., and can solve problems such as non-specificity

Inactive Publication Date: 2002-05-29
CANBREAL THERODIAGNOSTICS CANADA HLDG CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Diagnosis based on this serum enzyme measurement is therefore not specific

Method used

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Examples

Experimental program
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Effect test

example 1

[0060] Example 1: Isolation and purification of cell fluid NDP-Kinase from HL60 cells

[0061] The method for purifying cytosol NDP-Kinase from HL60 cells is described by Pulido-Cejudo et al. (FASEB J3: 608a, 1989). Briefly, this cell extract fraction was obtained from washing HL60 cell microparticles with 15 g (wet weight) of PBS buffer and applying it to a phosphate glucose column (P11) (1.6 cm x 28.0 cm), which had been previously buffered with phosphate buffer. solution (50mM potassium phosphate pH7.0, 2mM MgCl 2 , 1mMPMSF, 1mMDTT and 10% glycerol). Balanced. Wash with 300ml of the same buffer at a flow rate of 0.1ml / min. Unbound protein was concentrated into 10 ml by high-speed centrifugation, and YM5 membrane (5000 M.W. cutoff, AmiconDiv., Danvers, MA., USA) was used for high-speed centrifugation. The concentrate was added to a DEAE dextran column (2.6cm×28.5cm), which was pre-washed with Tris-buffer (50mM Tris-HCl pH7.5; 2Mm MgCl 2 ; 1 mM PMSF; 1 mM DTT and 10% (v / ...

example 2

[0066] Example 2: Separation and purification of es-LAPase

[0067]Breast cancer blasts from human tumor tissue were stimulated with 100nm 17-B-Estradiol for 24 hours, or culture medium was used as control. The cell medium is: RPMI 1640 culture medium + 10% FCS + 100U / ml penicillin + 100ug / ml streptomycin. Supernatants were collected after digestion with PBS in seamless dextran tubes (MV 12,400) for 24 hours (4°C). LAP was then purified from the degraded cell supernatant using HPLC-gel permeation chromatography with DEAE-cellulose and Bestatin-Sepharose affinity chromatography. Briefly, the cell supernatant was poured onto a Bio-Sil SEC-250 column (600×7.5 mm) containing 100 mM sodium phosphate (pH 6.8), 100 mM Na 2 SO 4 , 1uM ZnCl 2 , and 10% glycerol buffer pre-equilibrated. The column was then washed with 300ml of the same buffer at a flow rate of 0.5ml / min. The protein was concentrated to 10ml by high-speed centrifugation with YM5 membrane. The concentrated solution...

example 3

[0068] Example 3: Production and purification of monoclonal antibodies MAb 7B6 (anti-LAPase) and MAb 4A12 (anti-NDP-Kinase)

[0069] Methods in this protocol such as the preparation of immunizing antigens, the preparation of spleen cells from immunized animals, the fusion of spleen cells and myeloma cells, and the plating of fused cells in selective HAT medium were all derived from those described by Campbell and Lietzke and Unsicker. method described in detail. Unlike the standard method, our method for producing monoclonal antibodies is to use highly pure NDP-Kinase (7000-fold purity - see Table 1) for the first immunization instead of crude extracts and partially purified enzyme preparations. Ascites fluid was prepared by injecting 3 x 10 6 Myeloma cells were obtained by priming BALB / C mice with 500 l of norphytane one week before. Ascitic fluid was collected after 20 days. IgG immunoglobulins were purified from ascitic fluid by affinity chromatography on a 3 ml Protein ...

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Abstract

The identification and characterization of risk factors and their molecular implications in the pathophysiology of human diseases such as cancer is essential for designing efficient diagnostic assays and therapeutic compounds. Estrogenic steroids, under normal physiological conditions, have been shown to play a critical function in several tissues. The response of such a variety of tissues to estrogen stimulation can explain in part its active role in the development and progression of different human diseases, particularly breast cancer. Searching for estrogen-responding cellular factors in parental cells of primary human breast carcinomas obtained from tumour biopsies, two cellular markers, an isoenzyme of putative leucine aminopeptidase (LAPase; EC 3.4.11.1) from parental cells of primary human breast carcinomas obtained from tumour biopsies, and cytosolic NDP-Kinase / Nm23 (EC 2.7.4.6) from HL60 cells were identified. Monoclonal antibodies against each cellular marker have been produced. Determination of the presence of these two markers, either alone or in combination, -can be used to detect breast cancer, and in particular, associated metastatis. Thus, this invention refers to the use of both LAP and NDP-Kinase / Nm23 monoclonal antibodies together in a tandem solid-matrix based immuno-assay for first line confimatory blood-based testing for breast cancer.

Description

[0001] The content of the project is to use antibodies to detect specific methods of cancer. Specifically, the research on NDP-kinase / Nm23 and estrogen-stimulated leucine aminotyrase (es-LAPase) specific antibody and its application in the diagnosis of breast cancer and metastases. These two antibodies can also be used to detect NDP-kinase and LAPase levels in serum, plasma and living tissues. Background of the invention [0002] The key to effective detection and design of therapeutic drugs for human diseases such as breast cancer is the ability to identify risk factors and pathophysiological changes. Accurate diagnosis of the type and extent of cancer is key to selecting the best treatment options. The extent of the disease depends on the comprehensive analysis of tumor histology, blood component determination and cell marker detection. [0003] Widely used diagnostic methods for breast cancer include imaging techniques, such as ultrasound and X-ray methods. However, this...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/06A61K38/44A61K39/395A61P31/18C07K16/30C07K16/40C12N5/20C12Q1/37G01N33/569G01N33/573G01N33/574
CPCA61K39/395Y10S435/975G01N33/573G01N33/56988G01N33/57415A61K38/4813C07K16/40A61K2039/505C07K16/3015C12Q1/37A61P31/18A61K2300/00
Inventor 卡布里耶·普利多-塞胡多
Owner CANBREAL THERODIAGNOSTICS CANADA HLDG CORP
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