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A composite preparation of novel coronavirus rbd protein fluorescent microspheres

A technology of composite preparations and fluorescent microspheres, which is applied in the field of saturated competition, can solve the problems of inability to perform detection and affect the performance of detection reagents, and achieve the effect of maintaining antigen activity and improving detection sensitivity

Active Publication Date: 2022-04-08
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The stability of the fluorescent microspheres plays a key role in the subsequent detection steps. If the fluorescent microspheres are not properly stored, subsequent detection will not be possible, which will affect the performance of the final detection reagent.

Method used

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  • A composite preparation of novel coronavirus rbd protein fluorescent microspheres
  • A composite preparation of novel coronavirus rbd protein fluorescent microspheres
  • A composite preparation of novel coronavirus rbd protein fluorescent microspheres

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] A new crown RBD protein fluorescent microsphere composite preparation, the preparation method is as follows:

[0053] (1) Weigh 10mg ovalbumin and add it to phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4), then add 10-50mg / mL 1-ethyl-(3-dimethylaminopropyl ) Carbodiimide (EDC), avoid light reaction for 15-30min;

[0054] (2) Weigh 10 mg of bovine serum albumin and 10 mg of hemocyanin into the activated ovalbumin solution;

[0055] (3) Dialyze with phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4) to remove EDC;

[0056] (4) After affinity purification, the product whose molecular weight was the sum of the molecular weights of the three proteins was collected to obtain coupling protein 1 (BSA-OVA-KLH);

[0057] (5) Weigh 2.5g trehalose, 0.9g NaCl, 0.05g PEG4000, 0.05g Tween 20, 0.05gproclin 300, 0.24g Tris, 100g process water, add coupling protein 1 to make the final concentration 1%, adjust the pH to 8.5, to obtain the compound preparation.

Embodiment 2

[0059] A new crown RBD protein fluorescent microsphere composite preparation, the preparation method is as follows:

[0060] (1) Weigh 10mg bovine serum albumin and add it to phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4), then add 10-50mg / mL 1-ethyl-(3-dimethylaminopropyl base) carbodiimide (EDC), avoid light reaction for 15-30min;

[0061] (2) Weigh 10 mg of silk fibroin and add it to the activated bovine serum albumin solution;

[0062] (3) Dialyze with phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4) to remove EDC;

[0063] (4) After affinity purification, the product whose molecular weight was the sum of the molecular weights of the two proteins was collected to obtain coupling protein 2 (BSA-SF).

[0064] (5) Weigh 2.5g trehalose, 0.9g NaCl, 0.05g PEG4000, 0.05g Tween 20, 0.05gproclin 300, 0.24g Tris, 100g process water, add coupling protein 2 to make the final concentration 1%, adjust the pH to 8.5, to obtain the compound preparation.

Embodiment 3

[0066] A new crown RBD protein fluorescent microsphere composite preparation, the preparation method is as follows:

[0067] (1) Weigh 10mg of polylysine and add it to phosphate buffer (0.02-0.1M PBS, pH 7.2-7.4), then add 10-50mg / mL 1-ethyl-(3-dimethylaminopropyl base) carbodiimide (EDC), avoid light reaction for 15-30min;

[0068] (2) Weigh 10 mg bovine serum albumin and add it to the activated polylysine solution;

[0069] (3) Dialyze with phosphate buffered saline (0.02-0.1M PBS, pH 7.2-7.4) to remove EDC;

[0070] (4) After affinity purification, the product with higher protein concentration was collected to obtain coupling protein 3 (ε-PL-BSA);

[0071] (5) Weigh 5g sucrose, 1.3g MgCl 2 , 0.05g PEG4000, 0.02g Tetronin1307, 0.02g KroVin300M, 0.24g Tris, 100g process water, add coupling protein 3 to make the final concentration 1%, and adjust the pH to 8.5 to obtain a compound preparation.

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Abstract

The invention provides a composite preparation of novel coronavirus RBD protein fluorescent microspheres. The composite preparation is made of raw materials including the following mass percentages: 0.5-1% coupling protein, 2.5-5% sugar, 0.9-1.3% ion, large Molecular substance 0.05‑0.07%, surfactant 0.05‑0.1%, buffer 0.24‑1.21%, preservative 0.02‑0.05%. The new crown RBD protein fluorescent microsphere composite preparation of the present invention can be used for the preservation, dilution and treatment of the new crown RBD protein fluorescent complex and biological specimens. The saturation competition method detects neutralizing antibodies in human serum, plasma and whole blood samples, and the time-resolved fluorescent microsphere complex coupled with the new crown RBD protein needs to be preserved separately. The complex preparation can make the fluorescent microsphere complex not Generate aggregated precipitates, maintain antigen activity, and improve detection sensitivity. The biological samples include serum and plasma.

Description

technical field [0001] The invention belongs to the field of saturated competition, and in particular relates to a composite preparation of novel coronavirus RBD protein fluorescent microspheres. Background technique [0002] In the direct competition method in immunology, the labeled antigen and the antigen to be tested are both in liquid phase, and the chance of binding to the antibody is the same, and the relative binding rate of the labeled antigen is 50%. In the saturation competition method, the contact area between the solid-phase antigen and the antibody is small, and only the remaining antibody that binds to the antigen to be tested will bind to the solid-phase antigen, and the relative binding rate of the solid-phase antigen is 0%. Therefore, the slope of the inhibition curve of the indirect method will be greater than that of the direct competition method. The high sensitivity of the saturation competition method is sometimes difficult to achieve in ELISA, and th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/533
CPCG01N33/533
Inventor 王丽娇彭洁张变强于迪盛长忠粟艳周泽奇
Owner DYNAMIKER BIOTECH TIANJIN
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