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Beta2-microglobulin detection kit and clinical application thereof

A microglobulin and reagent technology, applied in the field of medical immunology in vitro diagnosis, can solve the problems of hindering widespread use, complicated operation and high cost, and achieve the effects of convenient mass production, large expression amount and simple operation.

Active Publication Date: 2021-11-09
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the disadvantages of cumbersome operation, long cycle or high cost, these methods hinder their widespread clinical use.

Method used

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  • Beta2-microglobulin detection kit and clinical application thereof
  • Beta2-microglobulin detection kit and clinical application thereof
  • Beta2-microglobulin detection kit and clinical application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Preparation of embodiment 1β2-MG mouse monoclonal antibody

[0036] 1. Animal immunization: Immune BALB / c female mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd.). For specific immunization conditions, please refer to the "Experimental Guidelines for Antibody Preparation and Use". The serum titer of immunized mice was tracked by indirect ELISA method, and the immunized mouse with the highest serum titer was selected for fusion experiment of mouse splenocytes and mouse myeloma cells.

[0037] 2. Cell Fusion

[0038] (1). Preparation of spleen cells

[0039] Take the immunized mice, remove their eyeballs, take blood, put them to death by breaking the cervical spine, soak them in 75% (v / v) alcohol for 10 minutes, take out their spleens in a sterile operating table, place them in a cell mesh, and grind the cells fully , passed through a sieve, and after being centrifuged and washed several times with sterile 1640 medium (purchased from Gibco), the cel...

Embodiment 2

[0049] Example 2. Recombinant expression and purification of single-chain antibody

[0050] According to the sequencing results in Example 2, a connecting peptide (GGGGS) 3 was added between the antibody heavy chain and light chain variable regions of the hybridoma cell lines BM15, BM18, BM20, BM22 and BM23, six histidines were introduced, and The entire gene was fused directly and the expression system of Pichia pastoris was used for recombinant expression of the single-chain antibody. The expressed antibodies were named as antibodies BM15, BM18, BM20, BM22 and BM23, respectively. The recombinant expression of the above-mentioned single-chain antibody is specifically as follows:

[0051] a) Construction of single-chain antibody gene expression vector

[0052] The gene sequence of the single-chain antibody BM15 is shown in SEQ ID NO:12, and the amino acid sequence is shown in SEQ ID NO:11; the gene sequence of the single-chain antibody BM18 is shown in SEQ ID NO:22, and the ...

Embodiment 3

[0064] Embodiment 3 The preparation and use method of detection kit

[0065] The detection kit of the present invention is a liquid dual reagent, including R1 reagent, R2 reagent, and calibrator, as follows:

[0066] 1. Reagent R1: Add stabilizer (for example: NaCl or KCl), coagulant (for example: PEG6000 or PEG8000), protective agent (for example: BSA or casein), preservative (for example: Proclin 300) to buffer 1 ), chelating agent (for example: EDTA), stir and mix well, that is the R1 reagent.

[0067] 2. R2 reagent: 5 kinds of recombinant anti-human β 2 -Dilute the antibody to 1.0mg / mL with phosphate buffer solution to prepare the antibody working solution; use MES buffer solution to dilute the latex microspheres (polystyrene latex, purchased from Japan JSR Company, particle size 80- 200nm) to 1% (mass concentration), then add 4% (mass ratio of EDC / latex microspheres) of EDC, stir and react at 30°C for 30min, remove unreacted EDC by centrifugation after the reaction, and...

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Abstract

The invention relates to five anti-human beta2-microglobulin (beta2-MG) antibodies and application thereof in a detection kit for detecting the content of human beta2-microglobulin in a blood sample. Specifically, the invention provides five mouse anti-human beta2-MG monoclonal antibodies and an application of the mouse anti-human beta2-MG monoclonal antibodies in a latex enhanced immunoturbidimetry detection kit. According to the invention, a plurality of antibodies are prepared and are paired and screened to obtain an antibody combination with sensitivity and specificity meeting requirements; meanwhile, the recombinant human beta2-MG is adopted as an antigen calibrator, so that the recombinant human beta2-MG can be conveniently and stably produced in batches with controllable quality, and the requirements of large-scale clinical application in the future can be met. A latex immunoturbidimetric quantitative detection reagent which is simple and convenient to operate and has sensitivity, specificity and related detection performance meeting human clinical sample detection is obtained by debugging and optimizing a detection system of the antibody combination.

Description

technical field [0001] The invention belongs to the field of in vitro diagnosis of medical immunity, especially a recombinant human β 2 - Microglobulin (β 2 -microglobulin, beta 2 -MG) and its role in the quantitative detection of human serum β 2 -Application in the detection kit of MG content. Background technique [0002] beta 2 - Microglobulin (β 2 -microglobulin, beta 2 -MG) is a protein with a molecular weight of 11.8kD, which exists in all nucleated cells (except erythrocytes and placental trophoblast), and is especially abundant in lymphocytes and monocytes. The daily production of β2-MG by the same individual remains constant and is secreted in various body fluids. Determination of beta in blood, urine, etc. 2 -MG is of great significance to the diagnosis of diseases, the following conditions will cause β in the blood 2 - Elevated MG content: ① Renal dysfunction: acute and chronic nephritis, renal function decline, renal tumors, etc., ② Malignant tumors: pri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/68G01N33/577G01N33/546
CPCC07K16/18G01N33/68G01N33/577G01N33/54313C07K2317/35C07K2317/56C07K2317/565G01N2333/4713Y02A50/30
Inventor 马永杨芸赵利利
Owner ZONHON BIOPHARMA INST
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