Hybridoma cell strain A4-1B1, procalcitonin monoclonal antibody generated by same, and application of procalcitonin monoclonal antibody

A technology of hybridoma cell line and calcitonin, which is applied in the field of monoclonal antibodies, can solve the problems of false positives, affect the level of clinical detection, low sensitivity of antibody detection, etc., and achieve the effect of high sensitivity

Inactive Publication Date: 2018-12-14
广东立诊得生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the PCT antibody in the current detection kits on the market is aimed at the entire PCT antigen, and the obtained PCT antibody may combine with CT in the serum, resulting in false positives, and the detection sensitivity of existing antibodies is low, which affects the clinical detection level

Method used

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  • Hybridoma cell strain A4-1B1, procalcitonin monoclonal antibody generated by same, and application of procalcitonin monoclonal antibody
  • Hybridoma cell strain A4-1B1, procalcitonin monoclonal antibody generated by same, and application of procalcitonin monoclonal antibody
  • Hybridoma cell strain A4-1B1, procalcitonin monoclonal antibody generated by same, and application of procalcitonin monoclonal antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Obtaining of PCT monoclonal antibody

[0020] Preparation of anti-PCT monoclonal antibody proceeds as follows:

[0021] 1. Preparation of immunogen: The protein sequence of PCT was obtained from NCBI, and the gene sequence was synthesized after prokaryotic expression codon optimization, connected to the prokaryotic expression vector pet41a, and expressed by IPTG induction. The cells were ultrasonically crushed and centrifuged to obtain the supernatant, which was passed through a nickel affinity column and a GST column, and the purified protein was used as an immunogen to immunize mice.

[0022] 2. Immunizing mice with antigens to prepare anti-PCT monoclonal antibodies, the specific implementation method is as follows: immunize mice with the above-mentioned purified protein PCT to prepare monoclonal antibodies. The specific steps are as follows: the purified immunogen was used to immunize the mice for the first time and two booster immunizations to prepare...

Embodiment 2

[0038] Example 2: Double-antibody sandwich method to screen the optimal PCT paired antibody and compare it with imported antibodies

[0039] We resuscitated the 127 monoclonal antibody cell lines we obtained, prepared monoclonal antibodies, purified them with octanoic acid method, labeled them with HRP, screened paired antibodies with checkerboard cross method, and screened out the best paired antibodies with clinical PCT positive mixed serum as the detection antigen. antibody pair.

[0040] The specific operation is as follows: coating antibody 6A2 and labeling antibody 1B1. Compared with commercial antibody pairs 16B3 (coated) / 42 (labeled) (purchased from Hytest Ltd), GRL1-2 (purchased from Grelin), NEM1-2 (purchased from Norman): Coating antibody 100 μl / well , the coating amount was 100ng, incubated at 37°C for 2h, and washed once with PBS-T; the clinical PCT positive mixed serum was diluted and added at 100ul / well, and a blank control well was set, incubated at 37°C for 3...

Embodiment 3

[0043] Embodiment 3: Western Blot identifies the antigen recognition site of the antibody

[0044] Divide PCT into 3 segments PCT-1 / 2 / 3, the corresponding amino acid sequence is as follows:

[0045] PCT-1: APFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASELEQEQEREGSSLDSPRSKR

[0046] PCT-2,CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP

[0047] PCT-3: GKKRDMSSDLERDHRPHVSMPQNAN

[0048] The hybridoma cell line 6A2 secretes antibodies to recognize the PCT-1 segment antigen recognition sequence, and the hybridoma cell line 1B1 secretes antibodies to recognize the PCT-3 segment antigen recognition sequence.

[0049] Connect the PCT3 segment PCT-1 / 2 / 3 to pet41a respectively, screen out the recombinant plasmids pet41a / PCT-1, pet41a / PCT-2, pet41a / PCT-3, and transform them into BL21(DE3)gold for prokaryotic expression; 3ml The expression bacteria were resuspended in 600μl lysate (20mM PB, pH7.8-8.0), and ice-bathed for 10min; the 0.3cm probe was ultrasonically broken, and the cycle was repeated 8 times (u...

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Abstract

The invention provides a hybridoma cell strain A4-1B1, a procalcitonin monoclonal antibody generated by the same, and an application of the procalcitonin monoclonal antibody. The hybridoma cell strainA4-1B1 is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO:C2017144. The procalcitonin monoclonal antibody can be applied to an immunoassay method. The present invention also provides a group of paired antibodies. The group of paired antibodies is composed of the antibody generated by the hybridoma cell strain A4-1B1 and an antibody generated by a hybridoma cell strain A4-6A2, and the hybridoma cell strain A4-6A2 is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO:C2017143. The antibodies provided by the invention have the advantages of high specificity, sensitiveness in reaction, and low cost, and can reflect the active state of infection. The anti-PCT monoclonal antibodies 1B1 and 6A2 are the optimal paired antibodies screened by a double-antibody sandwich method.

Description

technical field [0001] The invention relates to the field of monoclonal antibodies, in particular to hybridoma cell line A4-1B1 and the procalcitonin monoclonal antibody produced therefrom and applications thereof. Background technique [0002] Procalcitonin PCT is a protein. When the body suffers from severe bacteria, fungi, parasites or sepsis and multiple organ failure, the level of PCT in the plasma increases significantly. PCT has become a clinical systemic disease. One of the essential detection items in the diagnosis and treatment of bacterial infection. Under normal circumstances, neuroendocrine cells mainly limited to the thyroid and lung express and cleavage into calcitonin CT. Sepsis and pro-inflammatory cytokines can induce various types of cells in various tissues throughout the body to express and release PCT and enter the blood circulation system. Under the induction of cellular endotoxin, PCT can increase rapidly in 2-6 hours and reach the peak in 12-48 hou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/26G01N33/74G01N33/577
CPCG01N33/577G01N33/74C07K16/26G01N2333/585
Inventor 李小锋李晨阳杨伟民方雁毕景锐刘晓霞陈欣
Owner 广东立诊得生物科技有限公司
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