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A kind of sample pretreatment reagent and its application

A technology of samples and reagents, which is applied in the direction of analysis, immunoassay, and instruments through chemical reactions of materials, can solve problems such as biotin interference, and achieve strong affinity, reduce interference, and good accuracy

Active Publication Date: 2021-02-26
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the deficiencies and actual needs of the prior art, the present invention provides a sample pretreatment reagent and its application. The sample pretreatment reagent combines biotin in the sample in advance to solve the problem of biotin interference in immunoassays. The kit thus prepared has the advantages of good accuracy, fast detection speed, high sensitivity, and high degree of automation, which is conducive to providing a strong basis for rapid clinical diagnosis of fungal infection diseases

Method used

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  • A kind of sample pretreatment reagent and its application
  • A kind of sample pretreatment reagent and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The preparation of embodiment 1 kit

[0059] (1) Preparation of biotin-labeled capture antibody

[0060] The long-chain activated biotin was dissolved in dimethyl sulfoxide at a concentration of 1 mg / mL; the purified anti-Candida mannan monoclonal antibody (heavy chain is SEQ ID NO: 1, light chain is SEQ ID NO:2) was dissolved in 0.1mol / L sodium bicarbonate solution with pH=9.0 at a concentration of 1 mg / mL; the activated biotin solution was mixed with the antibody solution to be coupled at a ratio of 1:8, and warmed at room temperature Incubate for 4 hours; dialyze in 0.05 mol / L pH=7.2 PBS buffer at 4°C for 24 hours, and change the solution 4 times to remove unbound free biotin to obtain biotin-labeled capture antibody.

[0061] (2) Preparation of streptavidin-labeled magnetic particles

[0062] Resuspend the magnetic particles with 2-morpholineethanesulfonic acid buffer (MES) to make the concentration of the magnetic particles appropriate, add streptavidin to the ab...

Embodiment 2

[0065] Embodiment 2 One-step detection of candida mannan

[0066] This embodiment adopts one-step method to detect Candida mannan, and the steps are as follows:

[0067] (1) 10 μL of mannan with a concentration of 1 ng / mL and 150 μL of streptavidin-labeled magnetic particles with a concentration of 1 mg / mL (biotin load of 5000 pmol / mg) were co-incubated at 37 ° C for 10 min, and magnetically separated for 1 min. keep the supernatant;

[0068] (2) Add 150 μL of biotin-labeled capture antibody at a concentration of 0.3 μg / mL, 150 μL of streptavidin-labeled magnetic particles at a concentration of 0.3 mg / mL, and 150 μL of acridinium ester at a concentration of 0.3 μg / mL to the supernatant Label the signal antibody, mix well, incubate at 37°C for 10 minutes, magnetically separate for 1 minute, remove the supernatant, and wash 5 times with washing solution;

[0069] (3) Add 200 μL of excitation solution, mix thoroughly, and measure the chemiluminescence intensity of the solution ...

Embodiment 3

[0070] The preparation of embodiment 3 kits

[0071] (1) Preparation of biotin-labeled capture antibody

[0072] The long-chain activated biotin was dissolved in dimethyl sulfoxide at a concentration of 1 mg / mL; the purified anti-cryptococcal capsular polysaccharide monoclonal antibody (heavy chain is SEQ ID NO: 3, light chain is SEQ ID NO:4) was dissolved in 0.1mol / L sodium bicarbonate solution with pH=9.0 at a concentration of 1mg / mL; the activated biotin solution was mixed with the antibody solution to be coupled at a ratio of 1:8, and warmed at room temperature Incubate for 4 hours; dialyze in 0.05 mol / L pH=7.2 PBS buffer at 4°C for 24 hours, and change the solution 4 times to remove unbound free biotin to obtain biotin-labeled capture antibody.

[0073] (2) Preparation of acridinium ester-labeled signal antibody

[0074] Add an equal volume of 12.8mg / mL sodium periodate solution and 1% ethylene glycol solution successively to the acridinium ester solution, and react in ...

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Abstract

The present invention provides a sample pretreatment reagent and application thereof. The sample pretreatment reagent includes streptavidin magnetic particles and / or anti-free biotin antibody. In the present invention, in the detection process of the fungal antigen, in order to avoid the influence of biotin in the sample to be tested on the results, the sample to be tested is firstly pretreated with a sample pretreatment reagent to absorb and remove the biotin in the sample to be tested. After the biotin is primed, the antigen detection is carried out, which eliminates the interference caused by the biotin in the sample to be tested and significantly improves the accuracy of the detection.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnosis, and relates to a sample pretreatment reagent and its application, in particular to a sample pretreatment reagent for removing biotin interference, a chemiluminescence kit constructed therefor and its application. Background technique [0002] Magnetic particles refer to superparamagnetic materials with particle sizes ranging from nanometers to micrometers, and are often used as solid-phase carriers for immune reactions. The immunomagnetic particles formed by coating the antibody (or antigen) on the surface of the magnetic particles in a covalent or non-covalent manner can participate in the immune reaction, and the phase separation of the immune complex can be achieved by means of the superparamagnetism of the magnetic particles, which is convenient, Rapid in vitro diagnostics. [0003] Streptavidin is a tetrameric protein that has a strong affinity with small molecule biotin, and the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/543G01N21/76C07K16/14
CPCC07K16/14G01N21/76G01N33/54326G01N33/56961G01N2469/10
Inventor 刘春龙付彦凯张舟盛长忠周泽奇粟艳
Owner DYNAMIKER BIOTECH TIANJIN
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