Enzyme-linked immunosorbent assay kit for detecting concentration of tumor marker DKK1

An enzyme-linked immunosorbent reagent and tumor marker technology, applied in the field of tumor diagnosis, can solve the problems of expensive, unfavorable DKK1 large-scale census, expensive fluorescent markers, etc., achieve high specificity detection, improve the health of the whole people, relatively cheap detection Effect

Inactive Publication Date: 2016-05-18
FACON CO LTD
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  • Application Information

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Problems solved by technology

Chinese patent CN101398433 discloses a kit based on a time-resolved fluorescent immunoassay method, which requires a time-resolved fluorescent immunoassay analyzer that is not equipped in most hospitals, and this method also requires the use of relatively expensive fluorescent markers , is not conducive to a large-scale census of DKK1
Chinese patent CN102507951 discloses an enzyme-linked immunoassay kit for joint detection of tumor markers, which is coated with neuron-specific enolase (NSE), cytokeratin fragment 21-1 (CYFRA21-1) and Dkkopf1 (DKK1) multi-well plate of monoclonal antibody, horseradish peroxidase-labeled NSE, CYFRA21-1 and DKK1, chromogenic substrate TMB and substrate stop solution; each well of the multi-well plate is coated with NSE monoclonal antibody 1 -4ug/ml; each well is coated with CYFRA21-1 monoclonal antibody 1-2ug/ml; each well is coated with DKK1 monocl

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  • Enzyme-linked immunosorbent assay kit for detecting concentration of tumor marker DKK1
  • Enzyme-linked immunosorbent assay kit for detecting concentration of tumor marker DKK1
  • Enzyme-linked immunosorbent assay kit for detecting concentration of tumor marker DKK1

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Example Embodiment

[0020] Example 1:

[0021] based on figure 1 As shown in the schematic diagram of the principle of detection of DKK1 by enzyme-linked immunosorbent assay, the present invention provides an enzyme-linked immunoassay kit for detecting the concentration of tumor marker DKK1, which consists of a multi-well plate coated with DKK1 monoclonal antibody 1 and an enzyme-labeled DKK1 single Cloned antibody 2, chromogenic substrate (TMB) and substrate termination solution; among them, DKK1 monoclonal antibody 1 is 10C3; DKK1 monoclonal antibody 2 is 6E8; wherein the enzyme-labeled DKK1 monoclonal antibody 2 protects The solution is composed of 10 mM phosphate buffer with pH=7.2, 0.5% bovine serum albumin, 0.01% thimerosal and 50% glycerol; the storage condition of the enzyme-labeled DKK1 monoclonal antibody 2 is -20°C, and The concentration is greater than 100ug / ml; the working concentration dilution factor of the enzyme-labeled DKK1 monoclonal antibody 2 is 1:400, and the enzyme used in the...

Example Embodiment

[0022] Example 2:

[0023] A preparation method of an enzyme-linked immunoassay kit for detecting the concentration of tumor marker DKK1 specifically includes the following steps:

[0024] ①Prepare the multi-well plate coated with DKK1 monoclonal antibody 1: Dilute DKK1 monoclonal antibody 1 with pH=7-8 phosphate buffer, and then add the diluted DKK1 monoclonal antibody 1 solution to the wells of the multi-well plate Incubate at 37°C for 2 hours; after coating, wash the plate with phosphate buffer and pat dry; add 5% bovine serum albumin solution to each well of the multiwell plate, and block at 37°C for 1 hour; wash the plate with phosphate buffer after sealing Pat dry and store at 4°C. ②Preparation of enzyme-labeled DKK1 monoclonal antibody 2: Add 5mg / ml of HRP (horseradish peroxidase) to 0.2ml of 0.1M sodium periodate NaIO4 solution, and stir for 20 minutes at room temperature in the dark; 1mM pH4.4 sodium acetate buffer solution was dialyzed overnight at 4°C; after dialysis, ...

Example Embodiment

[0025] Example 3:

[0026] The present invention has extremely high sensitivity detection for DKK1: DKK1 standard substance is diluted gradually, namely 0.0125ng / ml, 0.0625ng / ml, 0.03125ng / ml, 0.015625ng / ml... Use this kit to measure its absorbance 5 times , And calculate the average and standard deviation. Simultaneously measure the DKK1 zero reference standard for 5 times, and calculate its average absorbance and standard deviation. The "t test" was used to test the significant difference between the diluted standard and the zero reference standard. The results are shown in Table 1. It has been determined that the detection limit of the present invention for DKK1 is less than 0.015625ng / ml (p <0.01).

[0027] Table 1

[0028] Concentration (ng / ml)

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Abstract

The invention discloses an enzyme-linked immunosorbent assay kit for detecting the concentration of the tumor marker DKK1. The enzyme-linked immunosorbent assay kit is composed of a porous plate coated with DKK1 monoclonal antibodies 1 with the model of 10C3, monoclonal antibodies 2 with the enzyme labeling model of 6E8, a chromogenic substrate and a substrate stop solution. The invention further discloses a preparation method and performance indexes of the enzyme-linked immunosorbent assay kit. The enzyme-linked immunosorbent assay kit matched with existing instruments in most hospitals is prepared by adopting the totally-domestic reagents, ultrahigh-sensitivity, high-specificity and low-cost detection of the concentration of DKK1 is achieved, large-scale general investigation of hepatocellular carcinoma is facilitated, and diagnosis can be carried out as early as possible to improve the health of all people of China.

Description

technical field [0001] The invention relates to the technical field of tumor diagnosis, in particular to an enzyme-linked immunoassay kit for detecting the concentration of a tumor marker DKK1. Background technique [0002] Worldwide, hepatocellular carcinoma (HCC) is the sixth most common malignancy and the third leading cause of cancer-related death. In my country, the high-risk population of hepatocellular carcinoma, including long-term carriers of various hepatitis viruses and patients with liver cirrhosis, is as high as 150 million. The 5-year overall survival rate of HCC is only 3-5%. Part of the reason for this extremely low survival rate is the lack of a timely and effective diagnosis, resulting in 60-70% of HCC being diagnosed at the time of diagnosis. Patients missed the best opportunity for treatment. [0003] The serum protein DKK1 discovered by the Shanghai Cancer Institute is a better marker than the existing hepatocellular carcinoma diagnostic marker alpha-f...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 王锋高鹏吴丹沈秋瑾覃文新
Owner FACON CO LTD
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