ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting prostate cancer

A prostate cancer and reagent kit technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of low prostate cancer specificity, elevated PSA, elevated prostate cancer, etc.

Active Publication Date: 2014-07-30
NAT RES INST FOR FAMILY PLANNING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The clinical early diagnosis method is mainly through the screening of prostate specific antigen (PSA). Up to now, PSA is the most mature and extensive serum tumor marker. However, in recent years, it has been found that PSA has obvious defects: in prostate Inflammation, urinary tract infection, catheterization, prostate massage, and cystoscopy can all lead to elevated PSA, with a large overlap, and the detection is affected by many factors
It can be seen that PSA is only specific to prostate tissue, but its specificity to prostate cancer is extremely low, and its increase can only indicate the possibility of prostate cancer

Method used

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  • ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting prostate cancer
  • ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting prostate cancer
  • ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting prostate cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Screening of Tumor Markers

[0023] 1. Experimental materials and equipment

[0024] 1. Main experimental materials

[0025] BPH-1 cells, LNCaP cells, PC-3 cells: purchased from the Cell Center of Peking Union Medical College; RPMI-1640 medium: GIBCO Company; fetal bovine serum: GIBCO Company; penicillin, streptomycin: Hyclone Company; trypsin: GIBCO Corporation; 25cm 2 Culture bottle, 75cm 2 Culture bottle: Corning Company; 15ml centrifuge tube: Corning Company; 5ml, 10ml pipettes: Corning Company; peroxyribonuclease I (DNAI): Shanghai Bioengineering Co., Ltd.; oxidized nucleoside complex (VRC), Leupetin, Aprotinin: Beijing Tianenze Gene Co., Ltd.; Immobiline pH gradient strips (Immobiline TM DryStrip, pH 3-10L, 24cm): GE Company; Protease Inhibitor Cocktail Set III: Calbiochem Company; PVDF membrane: Millipore Company; alkaline phosphatase-labeled goat anti-rabbit LaminA / C antibody was purchased from Pierce Company; Rabbit anti-human AnnexinA2 polyclon...

Embodiment 2

[0200] The assembly and use of embodiment two ELISA kits

[0201] 1. Experimental materials and instruments

[0202] 0.1M NaIO 4 , 1mM pH4.4 sodium acetate buffer, 0.2M pH9.5 carbonate buffer, NaBH4 solution, Tween-20, bovine serum albumin. Escherichia coli BL21, prokaryotic expression vector PET32.

[0203] Microplate reader (ELX800Universal Microplate Reader), shaker, electronic scale, stirrer, refrigerator.

[0204] 2. Experimental method

[0205] 1. Conjugation of Antibody to Horseradish Peroxidase

[0206] 1) Weigh 4mg horseradish peroxidase (HRP) and dissolve it in 1ml deionized water, add 200ul newly prepared 0.1M NaIO 4 , and lightly stirred at room temperature for 20 min in the dark until the solution was brownish green.

[0207] 2) Dialyze overnight at low temperature (4° C.) in 1 mol / L sodium acetate buffer solution with a pH of 4.4.

[0208] 3) Add 20 ul of 0.2M carbonate buffer solution dropwise to the above solution until the pH is 9-9.5, immediately add 5...

Embodiment 3

[0243] The clinical detection of embodiment three ELISA kits

[0244] Take 25 prostate cancer positive tissue samples and 25 prostate cancer negative samples, and use the kit and method described in Embodiment 2 to detect. The positive and negative specimens were confirmed by pathological examination of prostate cancer biopsy tissue.

[0245] Detected with HRP-labeled rabbit anti-human GRP78 monoclonal antibody, 21 of the 25 positive samples were positive, and 18 of the negative samples were negative.

[0246] Detected with HRP-labeled rabbit anti-human AnnexinA2 monoclonal antibody, 22 of the 25 positive samples were positive, and 20 of the negative samples were negative.

[0247] Using the prostate specific antigen kit (PSA kit), when PSA > 10ng / ml, 25 cases of prostate cancer patients were detected, the average of the three results was 15 cases of positive results, and 14 cases of negative samples were negative.

[0248] The accuracy rate of test kit of the present invent...

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Abstract

The compositions of three kinds of cell proteins of a benign prostatic hyperplasia (BPH-1) cell, an androgen dependence prostate cancer cell LNCaP and an androgen non-dependence prostate cancer cell PC-3 are compared by adopting a dimensional electrophoresis method. Through mass spectrum analysis and database retrieval, two difference proteins of GRP78 and Annexin A2 are determined. Through Western Blotting detection on the expression of the two difference proteins in the three kinds of cells, the GRP78 and the Annexin A2 are finally determined to be used as sign proteins for detecting the prostate cancer. The invention also discloses a preparation and a use methods of the ELISA kit for detecting the prostate cancer.

Description

Technical field: [0001] The invention relates to a detection kit, in particular to an ELISA kit capable of rapidly and effectively detecting prostate cancer. Background technique: [0002] In recent years, with the improvement of people's living standards, the aging of the population structure, and the change of diet structure, the incidence and mortality of prostate cancer (PCa) are on the rise, and the age of onset is becoming younger. Epidemiological data show that the incidence of prostate cancer in China has increased from 1.71 per 100,000 male population in 1993 to 7.9 per 100,000 male population in 2005, and it is increasing at a rate of 10% per year, becoming a threat to men's health. Main factors [ALVAREZA, LOKESHWAR V B. Bladder cancer biomarkers: current developments and future implementation. Curr Opin Urol, 2007, 17(5): 341-346.]. Due to the complex pathogenesis of PCa and its tendency to deteriorate and metastasize, most of them are diagnosed at the advanced o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/574
Inventor 王慧萍宁丽峰周越辛玲孙铁成于和鸣
Owner NAT RES INST FOR FAMILY PLANNING
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