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Method for covalently coupling amino-containing molecules to microspheres

A covalent coupling and amino group-containing technology, which is applied in the field of biochemical and clinical testing, can solve the problems of waste of raw materials, poor linearity of reagents, and affecting the measured value of samples to be tested.

Active Publication Date: 2013-09-18
BEIJING BEIJIAN XINCHUANGYUAN BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the phenomenon of self-aggregation will be relatively serious, which undoubtedly leads to waste of raw materials and increased costs
Moreover, the blank value of the prepared reagent is relatively high, and it is conceivable that this will inevitably damage the blank detection limit and sensitivity of the product.
In addition, the reagents prepared by the traditional one-step method have poor linearity when drawing the standard curve, which ultimately affects the measured value of the sample to be tested

Method used

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  • Method for covalently coupling amino-containing molecules to microspheres
  • Method for covalently coupling amino-containing molecules to microspheres
  • Method for covalently coupling amino-containing molecules to microspheres

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Traditional one-step conjugation of anti-Cys-C antibody

[0058] 1) Take 100 μL of 10% latex particles (150nm), add 2mL of 10mM MES solution (pH6.1) to mix, centrifuge at 15,000rpm for 50min, discard the supernatant, add 2mL of 10mM MES solution (pH6.1) and use a pipette to repeatedly suck The latex particles are dispersed and mixed evenly, and the concentration of the latex particles is 0.5%.

[0059] 2) Take 4.5 μL of rabbit anti-human Cys-C antibody with a concentration of 22 mg / mL, dilute it to 100 μL with PBS buffer solution of pH 7.4, and mix well.

[0060] 3) Mix the microsphere solution obtained in step 1) with 40 μL of the anti-Cys-C antibody solution obtained in step 2), place on a magnetic stirrer with a rotor and stir for 15 minutes to fully mix the two.

[0061] 4) Accurately weigh 0.0050 g of EDC and dissolve in 1 mL of deionized water to obtain 5 mg / mL of EDC, add 25 μL to the mixed solution of latex particles and anti-Cys-C antibody, and keep...

Embodiment 2

[0063] Embodiment 2: The method of the present invention (using Sulfo-NHS) coupling anti-Cys-C antibody

[0064] 1) Take 100 μL of 10% latex particles (150nm), add 2mL of 10mM MES solution (pH6.1) to mix, centrifuge at 15,000rpm for 50min, discard the supernatant, add 2mL of 10mM MES solution (pH6.1) and use a pipette to repeatedly suck The latex particles are dispersed and mixed evenly, and the concentration of the latex particles is 0.5%.

[0065] 2) Take 4.5 μL of rabbit anti-human Cys-C antibody with a concentration of 22 mg / mL, dilute it to 100 μL with PBS buffer solution of pH 7.4, and mix well.

[0066] 3) Mix the microsphere solution obtained in step 1) with 40 μL of the anti-Cys-C antibody solution obtained in step 2), place on a magnetic stirrer with a rotor and stir for 15 minutes to fully mix the two.

[0067] 4) Add 25 μL EDC (5 mg / mL) together with 25 μL Sulfo-NHS (50 mg / mL) to the mixed solution of latex particles and anti-Cys-C antibody at the same time, and k...

Embodiment 3

[0069] Embodiment 3: the preparation of Cys-C reagent

[0070] Get the latex particle of embodiment 1 (or 2) harvest and add 2mL0.01M PBS (pH7.4) ultrasonic dispersion and clean; NaN 3 , pH8.4) ultrasonic dispersion to obtain latex particles with a concentration of 0.2%; after magnetic stirring for 30 min, store at 2-8°C until use.

[0071] The formula is as follows:

[0072] 0.1M glycine buffer pH8.4;

[0073] 0.5% BSA;

[0074] 0.2% microspheres coupled with anti-Cys-C antibody;

[0075] 0.05%NaN 3 .

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Abstract

The invention relates to a method for covalently coupling amino-containing molecules to microspheres. According to the method, a reaction mixture is simultaneously in contact with carbodiimide and N-hydroxy succinimide (or N-hydroxy thiosuccimide)to carry out a covalent coupling reaction. A reagent prepared by utilizing the method disclosed by the invention can be used for effectively improving the self polymerization of the amino-containing molecules in a coupling process and has better linearity when a standard curve is drawn.

Description

technical field [0001] The present invention relates to the field of biochemical and clinical testing. In particular, the present invention relates to a method of covalent coupling. More specifically, it relates to a coupling method for covalently coupling amino-containing molecules to microspheres. Background technique [0002] In the traditional immunoturbidimetric method, if the concentration of the antigen or antibody to be detected in the sample is too low, or the molecular weight is too small, it is extremely difficult for the antigen-antibody complex to form turbidity unless it is left for a long time. If larger complexes are to be formed, the amount of antigen and antibody will increase accordingly. In view of the above defects, a latex-enhanced immune turbidimetric method has been developed. [0003] Latex-enhanced immunoturbidimetry uses biochemical methods to bind antibodies (or antigens) to microspheres. When the antigen (or antibody) to be detected in the sa...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/536
Inventor 姜敏
Owner BEIJING BEIJIAN XINCHUANGYUAN BIOLOGICAL TECH
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