Ratio immunoassay method with commercial magnetic beads as internal standard

An immune analysis and ratio technology, applied in the field of immune analysis, can solve the problems that hinder the wide application of SI, and achieve the effect of simple method, avoiding inaccuracy and improving accuracy

Active Publication Date: 2022-03-18
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite great success, complex IS labeling or synthesis procedures often hinder the widespread use of SI in bioquantitative analysis

Method used

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  • Ratio immunoassay method with commercial magnetic beads as internal standard
  • Ratio immunoassay method with commercial magnetic beads as internal standard
  • Ratio immunoassay method with commercial magnetic beads as internal standard

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] S1: Different amounts of CEA antibodies were incubated with 1 mL of AuNPs solution for 40 minutes.

[0051] S2: Measure the absorbance of the above mixed solution.

[0052] S3: the Ab 2 - After the AuNPs were purified by centrifugation, 300 μL of 3 M NaCl solution was added and mixed with it.

[0053] S4: Observe the color and state of the above solution.

[0054] Experimental results:

[0055] This experiment is to optimize the optimal dosage of the antibody modified gold nanoparticles of the present invention.

[0056] Wherein, the reaction conditions of this experiment are as described in Example 1.

[0057] Antibody dosages were designed to be 0, 1, 3, 5, 7, 10, 13, 15, 17, and 20 μg, respectively.

[0058] When the amount of added antibody is greater than 10 μg, Ab 2 - The absorbance of the AuNPs solution remains basically unchanged. This indicates that when the amount of antibody is about 10 μg, it is enough to complete the modification of AuNPs. And when ...

Embodiment 2

[0060] S1: Preparation of Ab 1 -MPs.

[0061] S2: Gold nanoparticles were incubated with 13 μg antibody for 40 minutes at room temperature to prepare Ab 2 -AuNPs, and blocked with BSA.

[0062] S3: Take 40 μL of diluted Ab 1 -MMP and 100 μL of fixed concentration of CEA were mixed evenly, and incubated with shaking at 37 ºC and 1500 rpm for a certain period of time.

[0063] S4: Wash the magnetic bead complex twice with 50 μL PBST solution. Then, add 50 μL of diluted Ab to the sample 2 -AuNPs, shake and incubate at 37 ºC, 1500 rpm for 1 h.

[0064] S5: Finally, after washing, the magnetic bead complex was reacted with 50 μL aqua regia for 20 min, and the solution was diluted to 8 mL with ultrapure water. Finally, the solution was collected by high-resolution inductively coupled plasma mass spectrometry 197 Au and 57 Fe signal and calculate the ratio of the two.

[0065] Experimental results:

[0066] This experiment is the Ab in the preferred invention 1 - Optimal r...

Embodiment 3

[0071] S1: Preparation of Ab 1 -MPs.

[0072] S2: Gold nanoparticles were incubated with 13 μg antibody for 40 minutes at room temperature to prepare Ab 2 -AuNPs, and blocked with BSA.

[0073] S3: Take 40 μL of diluted Ab 1 -MMP and 100 μL of fixed concentration of CEA were mixed evenly, and incubated at 37 ºC and 1500 rpm for 1 h with shaking.

[0074] S4: Wash the magnetic bead complex twice with 50 μL PBST solution. Then, add 50 μL of diluted Ab to the sample 2 -AuNPs, shake and incubate for a certain period of time under the conditions of 37 ºC and 1500 rpm.

[0075] S5: Finally, after washing, the magnetic bead complex was reacted with 50 μL aqua regia for 20 min, and the solution was diluted to 8 mL with ultrapure water. Finally, the solution was collected by high-resolution inductively coupled plasma mass spectrometry 197 Au and 57 Fe signal and calculate the ratio of the two.

[0076] Experimental results:

[0077] This experiment is the Ab in the preferred ...

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Abstract

The invention discloses a ratio immunoassay method with commercial magnetic beads as an internal standard. Carcino-embryonic antigen (CEA) is used as a research model, magnetic beads are used as carriers, and gold nanoparticles are used as reporter probes. And a 57Fe signal in the magnetic bead and a 197Au signal in the reporter probe are simultaneously acquired by adopting high-resolution inductively coupled plasma mass spectrometry (ICPMS). The 197Au/57Fe signal ratio and the CEA concentration have a good linear relationship within a certain range, so that accurate quantitative analysis of CEA is realized. The invention ingeniously develops another function (internal standard) of the carrier magnetic bead, and successfully avoids the magnetic bead loss in the magnetic separation process and the interference generated by the signal intensity fluctuation of the instrument, thereby enhancing the accuracy and reliability of the immunoassay method based on the magnetic bead. In consideration of the use universality of commercial magnetic beads, a new thought is possibly provided for a biological analysis method based on the magnetic beads.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the fields of immunoassay, ratio analysis method and stable isotope (Metal stable isotope) sensing. Background technique [0002] Magnetic beads (MPs) are usually made of γ-Fe 2 o 3 or Fe 3 o 4 Composed of microspheres, the size can range from nanometers to micrometers. The unique superparamagnetism avoids time-consuming and laborious separation procedures such as centrifugation and chromatographic separation, making magnetic microspheres the most widely used substrate for commercial use in the world. Magnetic beads have achieved great success in various biological applications such as cancer therapy, drug delivery, magnetic resonance imaging, blood purification, and molecular diagnostics. For example, near-infrared MPs imaging tracers with photothermal and magnetocaloric properties were developed based on magnetic microspheres and used for tumor ablation in mice. And created M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/543G01N33/553G01N27/62
CPCG01N33/57473G01N33/6848G01N33/54326G01N33/54346G01N33/553
Inventor 吕弋王超群刘睿
Owner SICHUAN UNIV
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