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Anti-human procalcitonin antibody and its application

A technology of human calcitonin and antibody, which is applied to anti-human procalcitonin antibody and its preparation. The application field of the above-mentioned antibody in the detection of human procalcitonin can solve the problem of poor stability of markers, failure to carry out, and increase in patients. burden and other issues, to achieve the effect of easy operation and convenient mass production

Active Publication Date: 2019-03-29
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can detect serum PCT of normal people, but it detects the mixture of free PCT, bound PCT and calcitonin gene-related peptide precursor, and cannot distinguish the above three substances; and the detection time is long (19-22h) , Polluted by radioactive elements, poor stability of markers, difficult to handle waste and other shortcomings, which limit its application
Chemiluminescence immunoassay (CLIA) generally needs to be equipped with expensive automatic electrochemiluminescence detectors, which cannot be carried out in conventional laboratories, and it brings unnecessary costs to patients and increases the burden on patients
The electrochemiluminescence immunoassay kit for detecting PCT using the biotin-avidin system also needs to be equipped with an expensive automatic electrochemiluminescence detector, which cannot be carried out in conventional laboratories, and it brings unnecessary costs to patients and increases the number of patients. burden

Method used

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  • Anti-human procalcitonin antibody and its application
  • Anti-human procalcitonin antibody and its application
  • Anti-human procalcitonin antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Preparation of anti-human procalcitonin hybridoma cell line

[0035] 1. Animal immunization

[0036] BALB / c female mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd.) were immunized with human recombinant procalcitonin 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.

[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 sie...

Embodiment 2

[0049] Example 2. Determination of the variable region sequence of the hybridoma cell line antibody

[0050] The sequence of the antibody variable region of the above-mentioned hybridoma cell line was determined.

[0051] a. Extraction of RNA: Extract the total RNA of the above-mentioned hybridoma cell line with reference to the instructions of the Total Cell RNA Extraction Kit (purchased from Roche Company) and perform reverse transcription immediately;

[0052] 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;

[0053] 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 variable regi...

Embodiment 3

[0056] Example 3. Recombinant expression and purification of single-chain antibody

[0057] 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 hybridoma cell strain antibody 3 , introduce six histidine tags (see SEQ ID NO: 9), and fuse the whole gene of histidine tags to perform codon optimization according to the preference of the Pichia pastoris expression system to perform recombinant expression of single-chain antibodies. Its structure is as attached figure 2 shown. The recombinant expression of the above-mentioned single-chain antibody is specifically as follows:

[0058] 1. Construction of expression plasmids for single-chain antibody genes

[0059] The nucleotide sequence of the codon-optimized single-chain antibody is shown in SEQ ID NO:10, and the amino acid sequence is shown in SEQ ID NO:11. The fragment of the optimized single-chain antibody whole gene synthesi...

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Abstract

The invention relates to an anti-human procalcitonin antibody and application thereof. A variety of antibodies are prepared, matched and screened, and an antibody combination with the sensitivity and specificity meeting requirements is obtained; meanwhile, mass production is convenient, and the requirement for large-scale clinic application in the future can be met. Debugging and optimization work of a detection system is carried out on the antibody combination, and a human PCT time-resolved immunofluorescence chromatography quantitative test card which is easy and convenient to operate is obtained, wherein the sensitivity, specificity and relevant detection performance can meet human clinic sample detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-human procalcitonin (PCT) antibody, a preparation method thereof and the application of the antibody in the detection of human procalcitonin. Background technique [0002] Procalcitonin (PCT) is a glycoprotein without hormone activity, composed of calcitonin, calcitonin, and N-terminal residue fragments, and is the precursor of calcitonin (CT). 13KDa, half-life 25-30 hours. Under physiological conditions, PCT is mainly produced by thyroid parafollicular C cells; under pathological conditions, PCT can be derived from various organs and tissues such as liver and lung. [0003] PCT has extensive and important application value in clinic. The plasma PCT content of healthy people is extremely low, lower than 0.5ng / ml in normal human blood. When severe bacterial, fungal, and parasitic infections occur, as well as sepsis and multiple organ failure, its level in plasma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/26C12N15/13C12P21/00G01N33/74
CPCC07K16/26C07K2317/56C07K2317/565G01N33/74G01N2333/5753
Inventor 马永赵利利王安良范宇
Owner ZONHON BIOPHARMA INST
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