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Method for amplifying antibody marking signal or nucleic acid probe marking signal

A signal amplification and antibody labeling technology, applied in the field of immunoassay, can solve problems such as low sensitivity, and achieve the effects of high sensitivity, good stability and no radioactive contamination.

Inactive Publication Date: 2013-08-07
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection sensitivity of radionuclide labeling is the highest, up to pg level, and the sensitivity of other methods is 1 to 3 orders of magnitude lower than that of radionuclide labeling

Method used

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  • Method for amplifying antibody marking signal or nucleic acid probe marking signal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Determination of carcinoembryonic antigen in human serum (taking 10 blood samples from the physical examination center of a hospital)

[0032] Preparation of LPS solution: Weigh 0.01g of LPS and dissolve in 1 mL of 0.1mol / L NaCO 3 Dissolve in the solution, then add 1 mL of newly prepared 0.1mol / L NaIO4 solution, after reacting in a water bath at 55°C for 120 minutes, add 40 μl of isopropanol, place in a refrigerator at 4°C for 30 minutes, centrifuge at 12,000 rpm / min for 10 minutes, discard the supernatant and wash the precipitate twice with 1 mL of pure water, each time 5 seconds, then with 1 mL of 0.1mol / L NaCO 3 solution to prepare LPS solution.

[0033] Preparation of antibody solution: take 0.01 g of rabbit anti-mouse antibody to be labeled, and use 0.5 mL of 0.1 mol / L NaCO 3 The solution was dissolved to prepare an antibody solution.

[0034] Preparation of LPS-antibody cross-linked product: LPS solution and antibody solution were mixed at a volume ratio o...

Embodiment 2

[0044] Determination of carcinoembryonic antigen in human serum (taking 10 blood samples from the physical examination center of a hospital)

[0045] Preparation of (1,3)-β-D-glucan (abbreviated as; dextran in this example) solution: Weigh 0.01g of dextran and dissolve in 1 mL of 0.1mol / L NaCO 3 Dissolve in the solution, then add 1 mL of newly prepared 0.1mol / L NaIO 4 After reacting in a water bath at 55°C for 60 minutes, add 40 μl of isopropanol, place in a refrigerator at 4°C for 30 minutes, then centrifuge at 12,000 rcf for 10 minutes, discard the supernatant and wash the precipitate twice with 1 mL of pure water, each time 5 seconds, then use 1 mL of 0.1mol / L NaCO 3 solution to prepare a dextran solution.

[0046] Preparation of antibody solution: take 0.01 g of rabbit anti-mouse anti-antibody to be labeled, and use 0.5 mL of 0.1mol / L NaCO 3 Dissolve in the solution to prepare antibody solution.

[0047] Preparation of dextran-antibody cross-linked product: Mix dextr...

Embodiment 3

[0056] Detection of fetal DNA in maternal peripheral blood (take blood samples from healthy pregnant women in a hospital, a total of 3 people):

[0057] The microplate is a product of Nunc Company in Denmark, and the probe is synthesized by ABI Company in the United States. The probe sequence is: 5′A TTC TT C GGC AGC ATC TCT GC 3′ (3′ hydroxylation).

[0058] Preparation of LPS solution: Weigh 0.01g of LPS and dissolve in 1 mL of 0.1mol / L NaCO 3 Dissolve in the solution, then add 1 mL of newly prepared 0.1mol / L NaIO 4 After reacting in a water bath at 55°C for 60 minutes, add 40 microliters of isopropanol, place in a refrigerator at 4°C for 30 minutes, and then centrifuge at 12,000 rpm / min for 10 minutes. Discard the supernatant and wash the precipitate twice with 1 mL of pure water. Each time for 5 seconds, and then dissolved in 1 mL of 0.1mol / L NaCO3 solution to prepare LPS solution.

[0059] Preparation of probe solution: Take a tube (1 OD) of probe DNA to be labeled, a...

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Abstract

The invention discloses a method for amplifying antibody marking signal or nucleic acid probe marking signal, and the main scheme is that: lipopolysaccharide of gram negative bacteria and an antibody are crosslinked, or fungi (1,3)-beta-D-glucan of fungi cell wall and the antibody or a nucleic acid probe are crosslinked, and then tachypleus amebocyte lysate and the gram negative bacteria lipopolysaccharide or fungi cell wall (1,3)-beta-D-glucan which is crosslinked with the antibody are specifically reacted, thereby improving the sensitivity of the antibody or nucleic acid probe detection. The system for amplifying antibody marking signal or nucleic acid probe marking signal can detect object substrates lower than pg level content.

Description

technical field [0001] The invention relates to a method for amplifying antibody labeling signal or nucleic acid probe labeling signal, that is, lipopolysaccharide (LPS) of Gram-negative bacteria or fungal (1,3)-β of fungal cell wall that specifically reacts with Limulus reagent - The D-glucan labeled antibody or nucleic acid is used for signal amplification in the immune analysis process and belongs to the technical field of immune analysis. Background technique [0002] The antibody amplification system is to mark highly sensitive and detectable substances that can react specifically to antibodies or antigen molecules. Through the reaction of the markers, the content of the detection substances is displayed, and at the same time, the signal of the detection is amplified through the enhancement of the signal. Improve the detection sensitivity of the substance to be detected. [0003] Current markers include fluorescein, enzymes (chromogenic substrates for chromogenicity or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
Inventor 孟春王航
Owner FUZHOU UNIV
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