Elisa kit for detecting early breast cancer and method for making same

An early-stage enzyme-linked immunosorbent assay technology, applied in the fields of molecular biology and cellular immunology, can solve the problems of complex sample processing equipment and other problems, and achieve the effects of shortening detection time, improving stability and convenient detection.

Active Publication Date: 2008-09-10
BEIJING MOKOBIO LIFE SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this method needs to obtain living tissue and requires complicated sample processing and expensive equipment

Method used

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  • Elisa kit for detecting early breast cancer and method for making same
  • Elisa kit for detecting early breast cancer and method for making same
  • Elisa kit for detecting early breast cancer and method for making same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of hGTA antigen

[0039] 1. Reverse transcription of human hGTA cDNA and construction of expression plasmid

[0040] ①Extraction of total RNA from breast cancer cells

[0041] Breast cancer cells MDA-MB453 (Breast Center, The First Affiliated Hospital of Harbin Medical University) were cultured in complete RPMI 1640 medium at 37°C, 5% CO 2 , cultured to the logarithmic growth phase under saturated humidity, digest the cells, pipet the cells, prepare the cell suspension, count, 2000r / min, discard the culture medium, suck the cells (5-10×10 6) in 1 mL of TRIZOL Reagent and placed at room temperature for 5 min to completely separate the nucleic acid-protein complex, centrifuged at 12,000 r / min for 5 min, took the supernatant, transferred it to a new RNase-free centrifuge tube, added 0.3 mL of chloroform, and shaken vigorously for 20 seconds. Place at room temperature for 5min, centrifuge at 13000r / min for 5min, the sample will be divided into three ...

Embodiment 2

[0076] Example 2 Preparation of anti-hGTA monoclonal antibody

[0077] 1) Preparation of monoclonal antibody against ALB antigen

[0078] 1. Test material:

[0079] 1) Human hGTA antigen: prepared in Example 1.

[0080] 2), experimental animals: six-week-old BALB / c mice were purchased from the Animal Experiment Center of Harbin Medical University;

[0081] 3), DMEM sugar medium: Hclone company

[0082] 4), other reagents: Freund's complete adjuvant, prepared by yourself

[0083] 2. Preparation method:

[0084] 1) Immunized animals:

[0085] Eight-week-old female BALB / C mice were immunized by intraperitoneal injection with an equal volume of human hGTA antigen and Freund's complete adjuvant, once a week, at a dose of 100ug / mice, and each mouse was intraperitoneally injected with 0.5ml. Then, the mice were immunized twice consecutively at an interval of 1 week. The dose and immunization method were the same. The adjuvant was changed to incomplete adjuvant. The antigen was ...

Embodiment 3

[0094] Example 3 Enzymatic labeling of anti-hGTA monoclonal antibodies

[0095] Weigh 2mg HRP and dissolve it in 1ml ultrapure water, add 30ul freshly prepared 0.1M NaIO 4 Put the above solution into a dialysis bag with an interception molecular weight of 8000, and dialyze the sodium acetate buffer of 2mM pH4.4 overnight; add 30 μl of 0.2M PH9.5 carbonate buffer to adjust the pH of the hydroformylation HRP to increase When it reaches 9.0~9.5, immediately add 0.5ml of anti-hGTA antibody, shake gently on a destaining shaker for 2 hours at room temperature in the dark; add 40ul of newly prepared 4mg / ml NaBH 4 solution, mix well, and set at 4°C for 2 hours. The above-mentioned solution was put into a dialysis bag with a molecular weight of 8000, dialyzed against 0.01M PH8.2 phosphate buffer overnight, and purified by the ammonium sulfate method.

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Abstract

The invention discloses an ELISA kit for detecting early breast cancer in vitro and a preparation method. The ELISA kit comprises an enzyme label plate covered with anti-hGTA monoclonal antibody, enzyme label resistance anti-hGTA monoclonal antibody, recombinant human hGTA standard, concentrated washing solution, concentrated diluent, stopping solution and enzyme substrate solution, wherein, the hGTA is an amino acid sequence showed in SEQ ID NO: 2. Manifested by clinic application experiments, early breast cancer can be accurately detected by the ELISA kit of the invention which has the advantages of strong specificity, high sensitivity, long period of validity and low cost, etc. and can be applied to the clinical detection of breast cancer.

Description

technical field [0001] The invention relates to an in vitro detection kit for tumors, in particular to an enzyme-linked immunosorbent assay kit for in vitro detection of early breast cancer and a preparation method thereof, belonging to the fields of molecular biology and cellular immunology. Background technique [0002] Breast cancer is the most common malignant tumor in women. Even in developed countries, breast cancer is one of the leading causes of death among women, with an annual incidence of more than 70 / 100,000. [0003] There are many markers of breast cancer, but it is generally believed that tumor markers have no practical value in the early diagnosis of breast cancer, nor can they be used for breast cancer screening in a large population. Soon after the first tumor marker CA15-3 in breast cancer was established, it was seen that in stages I and II of breast cancer, only about 10% of patients had elevated serum CA15-3 levels, and some other effective Tumor marke...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/574
Inventor 金鑫王文雅戴路杜军
Owner BEIJING MOKOBIO LIFE SCI CO LTD
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