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Anti-human myoglobin antibody and its application in detection kit

A human myoglobin and antibody technology, applied in the direction of anti-animal/human immunoglobulin, application, biological testing, etc. The effect of convenient mass production

Active Publication Date: 2020-09-22
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Colloidal gold immunochromatography has the advantages of simple and fast operation, single detection, easy storage, and no need for special equipment. However, the disadvantages of low sensitivity and difficulty in quantification limit the use of this method.
Enzyme-linked immunoassay has the characteristics of simple operation, long validity period of reagents, no pollution, higher sensitivity than colloidal gold, good specificity, and the results can be measured by instruments. However, due to its relatively low sensitivity, the labeling enzyme and substrate used The shortcomings of narrow quantitative measurement range and narrow instrument measurement range limit its application in microimmunoquantitative assays
Although chemiluminescence immunoassay has the advantages of accuracy, high sensitivity, strong specificity, and good precision, the instruments used are expensive and the reagents used are imported reagents, which are difficult to carry out in general laboratories and are not suitable for emergency testing.

Method used

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  • Anti-human myoglobin antibody and its application in detection kit
  • Anti-human myoglobin antibody and its application in detection kit
  • Anti-human myoglobin antibody and its application in detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1. Preparation of anti-human MYO hybridoma cell line

[0042] 1. Animal immunization

[0043]BALB / c female mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd.) were immunized with recombinant human MYO (recombinantly expressed in Escherichia coli, produced by our company) according to the general immunization procedure. 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.

[0044] 2. Cell Fusion

[0045] (1). Preparation of spleen cells

[0046] 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 me...

Embodiment 2

[0056] Example 2. Determination of the variable region sequence of the hybridoma cell line antibody

[0057] The sequences of the antibody variable regions of the hybridoma cell lines M08 and M26 were determined.

[0058] a. Extraction of RNA: Extract the total RNA of the above-mentioned hybridoma cell lines M08 and M26 with reference to the instructions of the Total Cell RNA Extraction Kit (purchased from Roche Company) and perform reverse transcription immediately;

[0059] b. Reverse transcription of RNA into DNA: refer to Thermo Scientific Reverted First strand cDNASynthesis Kit (purchased from Thermo Company) to reverse transcribe the total RNA extracted in the previous step to obtain cDNA, and freeze it at -20°C for later use;

[0060] c. PCR amplification and recovery of the variable region sequence: the cDNA obtained in the above step is used as a template, and the variable region sequence of the heavy chain and light chain is sequenced with the general primer for the ...

Embodiment 3

[0064] Example 3. Recombinant expression and purification of single-chain antibody

[0065] According to the sequencing results in Example 2, a connecting peptide (GGGGS) was added between the heavy chain and light chain variable regions of the M26 and M08 antibodies, respectively. 3 , and its whole gene was synthesized, and the expression vector construction and recombinant expression of the single-chain antibody were carried out. The recombinant expression of the above-mentioned single-chain antibody is specifically as follows:

[0066] a) Construction of single-chain antibody M26 and M08 expression plasmids

[0067] The nucleotide sequence of the single-chain antibody M26 is shown in SEQ ID NO: 19, the amino acid sequence is shown in SEQ ID NO: 17, the nucleotide sequence of the single-chain antibody M08 is shown in SEQ ID NO: 20, and the amino acid sequence is shown in SEQ ID NO:18 shown. The single-chain antibody M26 and M08 genes were introduced into the NcoI restrict...

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Abstract

The invention relates to a novel anti-human myoglobin antibody and an application thereof in a detection kit, and belongs to the field of immunochemistry. According to the invention, a plurality of antibodies are prepared, and paired screening is carried out to obtain an antibody combination (M26 and M08) which meets requirements in sensitivity and specificity. Meanwhile, the antibody combinationis convenient for mass production and can meet the requirement of future large-scale clinical application. The debugging optimization work of a detection system for the antibody combination is carriedout to completely meet the application requirements of preparing fluorescent rapid detection test paper cards for human myoglobin. The fluorescent rapid detection test paper cards are simple and convenient to operate, and accord with commercial application indexes in sensitivity, specificity and related detection performance.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to two anti-human myoglobin (MYO) antibodies, a preparation method thereof and the application of the antibodies in human myoglobin detection. Background technique [0002] Myoglobin (MYO) is an oxygen-binding protein specific for skeletal and cardiac muscle. The molecular weight is 16.7KDa, it is composed of a polypeptide chain containing 153 amino acid residues and a heme prosthetic group, and is in a compact spherical shape. [0003] Due to the small molecular weight of myoglobin, when muscle cells are damaged, myoglobin will be released into the circulatory system. It rises in 1-2 hours, reaches the peak in 12 hours, and gradually decreases after 24 hours. It is an early diagnosis of acute myocardial infarction and other diseases. important reference indicators. [0004] Currently, immunoassay methods for myoglobin mainly include colloidal gold immunochromatography, en...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/70C12N1/21G01N33/68
Inventor 马永赵利利赵百学
Owner ZONHON BIOPHARMA INST
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