TRFIA for synchronously detecting AFP and AFP-IgM and reagent kit thereof

A synchronous detection and kit technology, applied in the field of biochemical detection, can solve problems such as cumbersomeness, affecting diagnostic efficiency, and complex detection process.

Inactive Publication Date: 2010-06-23
RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the technical problem to be solved by the present invention is to aim at the defects that in the existing methods, the tumor marker AFP and the auxiliary marker AFP-IgM of HCC are detected separately, the detection process is relatively complicated and cumbersome, and the diagnostic

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  • TRFIA for synchronously detecting AFP and AFP-IgM and reagent kit thereof
  • TRFIA for synchronously detecting AFP and AFP-IgM and reagent kit thereof
  • TRFIA for synchronously detecting AFP and AFP-IgM and reagent kit thereof

Examples

Experimental program
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Embodiment 1

[0043] The preparation of embodiment 1 kit

[0044] The reagents in each box are sufficient for 96 measurements, and the materials in the box are as follows:

[0045] (1) 1 x 96-well plate (8 strips x 12 wells, which can be split into single wells) is coated with anti-mouse anti-human AFP monoclonal antibody.

[0046] (2) 6×AFP / AFP-IgM mixed standard, lyophilized product. Dissolve each bottle with 1ml of distilled water before use.

[0047] (3) 1× europium-labeled antibody freeze-dried product, dissolved in 0.5ml distilled water before use.

[0048] (4) 1× samarium-labeled antibody freeze-dried product, dissolved in 0.5ml distilled water before use.

[0049] (5) 1× buffer solution, 30ml.

[0050] (6) 1× washing solution, 30ml, dilute with distilled water at a volume ratio of 1:25 when used.

[0051] (7) 1× enhancement solution, 15ml.

[0052] The following are the specific ingredients contained in each material in the kit and their preparation methods.

[0053] 1. Micro...

Embodiment 2

[0071] The preparation of embodiment 2 kit

[0072] Materials in the kit: the same as in Example 1 except that the 96-well plate (1) coated with anti-mouse anti-human AFP monoclonal antibody is not included.

[0073] The preparation method of each material in the kit is as follows:

[0074] (1) Eu 3+ Preparation of mouse anti-human AFP monoclonal antibody by labeling

[0075] Refer to Eu 3+ Labeling kit (manufacturer PerkerElmer) manual operation. Specifically: Take 1 mg of mouse anti-human AFP monoclonal antibody and dissolve it in 0.25mol / L NaHCO 3 Add 0.5mg Eu to 200μl 3+ -N 2 -[p-isocyanate-benzyl]-diethylenetriaminetetraacetic acid (Eu 3+ -DTTA) and mix thoroughly, and react at 4°C for 24 hours. The reaction solution was chromatographed on a Sephadex G-50 column (1×20 cm), monitored by A280 to collect protein peaks, combined peak tubes, and determined the protein content. At the same time with PerkinElmer Eu 3+ Determination of Eu of combined peaks with standard...

Embodiment 3

[0079] The preparation of embodiment 3 kits

[0080] The reagents in each box are sufficient for 48 measurements, and the materials in the box are as follows:

[0081] (1) 1×48-well plate (4 strips×12 wells, which can be split into single wells) is coated with mouse anti-human AFP monoclonal antibody.

[0082] (2) 6×AFP / AFP-IgM mixed standard, lyophilized product. Dissolve each bottle with 1ml of distilled water before use.

[0083] (3) 1× europium-labeled antibody freeze-dried product, dissolved in 0.5ml distilled water before use.

[0084] (4) 1× samarium-labeled antibody freeze-dried product, dissolved in 0.5ml distilled water before use.

[0085] (5) 1× buffer solution, 30ml.

[0086] (6) 1× washing solution, 30ml, dilute with distilled water at a volume ratio of 1:25 when used.

[0087] (7) 1× enhancement solution, 15ml.

[0088] The preparation method of each material in the kit is as follows:

[0089] 1. Microwell reaction plate preparation

[0090] Use 50mmol / L...

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Abstract

The invention discloses a time resolution fluorescence immunity analysis (TRFIA) method for synchronously detecting AFP and AFP-IgM and a reagent kit thereof. The reagent kit comprises the following reagents: 1) an AFP monoclonal antibody of a first epitope; 2) an AFP monoclonal antibody of a second epitope marked by a lanthanide element; 3) an IgM monoclonal antibody marked by another lanthanide element; 4) AFP/AFP-IgM mixed standard products; 5) a buffer solution; 6) washing liquid; and 7) enhancing liquid, wherein the lanthanide elements are selected from europium and samarium, and the AFP monoclonal antibody of the second epitope and the AFP monoclonal antibody of the first epitope have different epitopes. The invention adopts the TRFIA with the high sensitivity, the method for synchronously detecting AFP and AFP-IgM is established, the and the invention has the advantages of high sensitivity, strong specificity, good stability and simple operation, can realize the high atomization, can improve the speed of the clinical examination, can greatly reduce the artificial error, can improve the reliability of the detected results, and greatly improves the diagnosis efficiency.

Description

technical field [0001] The invention belongs to the field of biochemical detection, in particular to a time-resolved fluorescent immunoassay method for synchronous detection of alpha-fetoprotein and alpha-fetoprotein-immunoglobulin complex and a kit thereof. Background technique [0002] Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide, ranking fourth in incidence. So far, there is no ideal highly sensitive and specific serum marker that can be used for the diagnosis of HCC, especially the early diagnosis. At present, the clinically commonly used alpha-fetoprotein (AFP), if the cutoff value is set at 20 ng / ml, the sensitivity for diagnosing liver cancer is only 60-80%, and it is only 40% for small liver cancer. Increased AFP also occurs in some benign liver lesions. For example, about 15-18% of patients with chronic hepatitis and 11-47% of patients with liver cirrhosis have serum AFP at a level of 20-200ng / ml, so it is difficult to diagn...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/574
Inventor 盛世乐黄钢王青
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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