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Method for preparing latex enhanced turbidimetric immunoassay reagent by using recombinant antibody

A recombinant antibody and latex-enhanced technology, which is applied in peptide preparation methods, botany equipment and methods, biochemical equipment and methods, etc. The efficiency is not easy to increase and other problems, to achieve the effect of reducing costs, avoiding unstable batch-to-batch differences, fast and stable production

Active Publication Date: 2022-06-24
WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims to provide a method for preparing latex-enhanced immunoturbidimetric reagents by using Fc-modified recombinant antibodies, and then on the one hand solve the problems of unstable batch-to-batch differences between polyclonal antibodies and monoclonal antibodies, huge manpower investment, and difficulty in improving antibody yields, etc. On the other hand, it effectively improves the coupling efficiency of the solid phase and the antibody in the reagent preparation process, which forms a favorable support for PETIA detection reagents to further reduce the cost and improve the quality

Method used

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  • Method for preparing latex enhanced turbidimetric immunoassay reagent by using recombinant antibody
  • Method for preparing latex enhanced turbidimetric immunoassay reagent by using recombinant antibody
  • Method for preparing latex enhanced turbidimetric immunoassay reagent by using recombinant antibody

Examples

Experimental program
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Embodiment 1

[0034] Example 1 Modification and expression of recombinant antibodies

[0035] (1) Modification strategy of recombinant antibody

[0036] The recombinant antibody is a recombinant product of mouse anti-Fab end and human IgG1 type Fc end, and between the CH2 and CH3 regions of the Fc end of the antibody, a short peptide of 10 amino acids is inserted: GGG GGGGGGG or KKK KKKKKKK or FFFF FFFFFF.

[0037] Before modification, the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2; As shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5.

[0038] (2) The mouse anti-Fab end and the Fc end of the human IGG1 antibody modified in step (1) are edited at the gene level, and the base sequences encoding the heavy chain and the light chain are obtained by gene synthesis.

[0039] (3) Using plasmid vector pCDNA3, the base sequence obtained in step (2) was introduced into mammalian cells (CHOS, C...

Embodiment 2

[0054] Example 2 The method for preparing latex-enhanced immune turbidimetric reagent R2 using the recombinant antibody prepared in Example 1

[0055] (1) Dilute 1 mL of carboxylate latex microspheres (50nm-400nm) with 4mL of 20-50mM Good'S buffer to a mass concentration of 1% carboxyl latex microspheres, and mix well at 25-37°C.

[0056] (2) Weigh 0.01g EDC·HCl (C 8 H 17 N 3 HCl), added to the latex microsphere solution obtained in step (1), and rapidly stirred at 25-37° C. to activate carboxyl groups. After 10-30 minutes of reaction, centrifuge (30000g, 10-60min), remove the supernatant, and collect the precipitate for future use.

[0057] (3) Use 5mL of 20-50mM Good'S buffer to resuspend the above pellet, and disperse by ultrasonic (40KHz, 5-15min).

[0058] (4) Add 2 mg of the recombinant antibody prepared in Example 1 to the latex microsphere solution that was rapidly stirred in step (3) at 25-37° C., respectively, to carry out the coupling reaction of the latex micro...

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Abstract

The invention discloses a method for preparing a latex enhanced immunoturbidimetric reagent by using a recombinant antibody, and belongs to the field of in-vitro diagnostic reagents. A segment of amino acid sequence is inserted between CH2 and CH3 at the Fc end of the antibody to obtain the recombinant antibody, so that the length of the Fc end is increased, the hydrophobicity of the Fc end is improved, and the Fc end is more easily coupled with latex microspheres. When the recombinant antibody is used for preparing a reaction reagent R2 for latex-enhanced immunoturbidimetry, the problems that the batch difference is unstable, the human input is huge and the antibody yield is not easy to increase due to the adoption of common polyclonal antibodies and monoclonal antibodies can be avoided.

Description

technical field [0001] The invention relates to a method for preparing a latex-enhanced immune turbidimetric reagent using recombinant antibodies, in particular to a method for preparing a latex-enhanced immune turbidimetry kit using recombinant antibodies modified at the Fc end, and belongs to the field of in vitro diagnostic reagents. Background technique [0002] Latex-enhanced immunoturbidimetric assay (PETIA) is a relatively stable and accurate method for homogeneous immunoturbidimetric detection of body fluid proteins that has emerged in recent years. The PETIA method is roughly divided into two types: nephelometric detection and transmission turbidimetric detection. The basic principles of these two types are very similar. Antibodies are conjugated on the surface of polymer latex microspheres. When the antibody-conjugated microspheres are combined with the antigen, they quickly aggregate together in a short period of time, changing the astigmatism properties of the re...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K1/107C12N15/13C12N15/85G01N33/68G01N33/543
CPCC07K16/18C12N15/85G01N33/68G01N33/54313G01N2333/4737Y02A50/30
Inventor 王亭芳
Owner WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
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