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A kind of procalcitonin monoclonal antibody and its application

A technology of monoclonal antibody and cloned antibody, applied in the direction of calcitonin, antibody mimetic/scaffold, hormone peptide, etc., can solve the problems of waste of human and financial resources, inability of monoclonal antibody, prolonging the preparation time of effective antibody, etc. High sensitivity, shortened preparation time, and labor-saving effect

Active Publication Date: 2019-03-12
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the whole PCT antigen is used to directly immunize mice, the finally selected cell line that can stably secrete PCT monoclonal antibody is likely to be stimulated by the same site on the antigen with strong immunogenicity to stimulate the body to produce, or it can only recognize Calcitonin or anti-calcitonin, so that the final monoclonal antibody cannot be used for subsequent applications, resulting in prolongation of effective antibody preparation time and waste of human and financial resources

Method used

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  • A kind of procalcitonin monoclonal antibody and its application
  • A kind of procalcitonin monoclonal antibody and its application
  • A kind of procalcitonin monoclonal antibody and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1: Synthesis of N-PCT-KLH and KLH-KC Immunogens

[0027] N-PCT-KLH and KLH-KC immunogens are obtained by coupling thiolated hemocyanin (KLH-SH) to artificially synthesized procalcitonin polypeptide fragments N-PCT and anticalcitonin (KC), respectively. The operation steps are as follows:

[0028] (1) Procalcitonin polypeptide fragment N-PCT

[0029] (APFRS ALESSPADPA TLSEDEARLL LAALVQDYVQ MKASELEQEQ EREGSSLDSP RS) and KC (DMSSDLERDH RPHVSMPQNA N) sequences were sent to Shanghai Jill Biological Company for synthesis, and the synthesis purity was >95%.

[0030] (2) Artificially synthesized N-PCT (6.8×10 3 Da) 3.2mg and anticalcin (2.4×10 3 Da) 1.2 mg was dissolved in 1 ml of 0.01M pH7.4 phosphate buffer to prepare a polypeptide solution. 1 mg crosslinker Sulfo-SMCC was dissolved in 500 μl deionized water. Put KLH-SH (4×10 5 Da) 5mg was dissolved in 1ml deionized water, shake gently to dissolve.

[0031] (3) Mix the Sulfo-SMCC solution with the two polypepti...

Embodiment 2

[0035] Example 2: Preparation of procalcitonin monoclonal antibody

[0036] N-PCT, KC polypeptide and the N-PCT-KLH and KLH-KC conjugates prepared above were used to inoculate male 6-7 week-old Balb / c mice respectively by conventional methods. For the first immunization, the immunogen was diluted to 1 mg / ml with 0.01M pH7.4 phosphate buffer solution, emulsified and mixed with complete Freund's adjuvant, 50 μg of the immunogen per mouse. Inoculate by subcutaneous injection in abdomen and back of mice. Every 2 to 3 weeks, the same amount of immunogen was emulsified and mixed with Freund's incomplete adjuvant for booster immunization, a total of 4 times of immunization, and 1 week after the last immunization, blood was collected from the orbit of the immunized mice, and the serum was separated by centrifugation. In the indirect ELISA method, the whole antigen of procalcitonin is used to coat the microtiter plate, and the immune titer is detected respectively. Results N-PCT, KC ...

Embodiment 3

[0038] Example 3: Specific identification of procalcitonin monoclonal antibody recognition

[0039] The specificity of procalcitonin monoclonal antibody was identified with 2 μg / ml of PCT, CT, CGRP and C-reactive protein. The ELISA method was used for detection, and PCT was used as a positive control, and the results were as follows: figure 1 Shown, CT, CGRP, C-reactive protein were all negative. It shows that the PCT monoclonal antibody of the present invention has high specificity.

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Abstract

The invention discloses a procalcitonin monoclonal antibody and application thereof. The antibody is produced by hybridoma cell secretion with a collection number of CGMCC NO: 12035. The procalcitonin monoclonal antibody provided by the invention has an effect of directionally identifying different epitope areas of the procalcitonin. The experiment proves that the procalcitonin monoclonal antibody provided by the invention can be combined with procalcitonin at high specificity and is high in sensitivity and stability. A detection reagent prepared from the procalcitonin antibody can be used for accurately measuring procalcitonin in serum, is high in sensitivity and accuracy, has high correlation with the detection results of available reagents in the market, has high diagnostic agreement with clinical diagnosis and can be used for clinical auxiliary diagnosis of sepsis or bacterial infection.

Description

technical field [0001] The invention relates to a procalcitonin monoclonal antibody, in particular to the preparation and application of a procalcitonin monoclonal antibody and a procalcitonin immunogen, and belongs to the field of biotechnology. The procalcitonin monoclonal antibody of the present invention is produced by the secretion of hybridoma cells with the number CGMCC NO: 12035 preserved by the General Microbiology Center of the China Microbiological Culture Collection Management Committee; the address of the preservation unit is: No. 1 Beichen West Road, Chaoyang District, Beijing No. 3 Institute of Microbiology, Chinese Academy of Sciences, Zip Code: 100101, Tel: 010-64807355, Fax: 010-64807288, Email: cgmcc@im.ac.cn ;The date of preservation is: January 28, 2016. Background technique [0002] PCT (procalcitonin) is a non-hormonally active calcitonin propeptide substance, which is encoded by the Calc-I gene located on chromosome 11. PCT is composed of 116 amino ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/26C07K14/585C07K19/00G01N33/74G01N33/577
CPCC07K14/585C07K16/26C07K2319/00G01N33/74G01N2333/585
Inventor 邹炳德邹继华赵金华祝快昌张吉燕
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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