Anti-human tissue kallikrein 1 antibody and its application

A kallikrein and antibody technology, applied in the field of immunochemistry, can solve the problem that specific antibodies cannot effectively recognize natural hK1, and achieve the effect of convenient mass production and easy operation

Active Publication Date: 2019-04-05
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When using recombinant hK1 for screening of specific antibodies, there will be a problem that the screened specific antibodies cannot effectively recognize natural hK1

Method used

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  • Anti-human tissue kallikrein 1 antibody and its application
  • Anti-human tissue kallikrein 1 antibody and its application
  • Anti-human tissue kallikrein 1 antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1. Preparation of anti-human tissue kallikrein 1 hybridoma cell line

[0054] 1. Animal immunization

[0055] BALB / c female mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd.) were immunized with recombinant human tissue kallikrein 1 (expressed in Chinese hamster ovary cells, see patent 201310746269.X for the preparation method) 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, and the fusion experiment of mouse splenocytes and mouse myeloma cells was carried out.

[0056] 2. Cell Fusion

[0057] (1). Preparation of spleen cells

[0058] 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,...

Embodiment 2

[0068] Example 2. Determination of the variable region sequence of the hybridoma cell line antibody

[0069] The variable region sequences of the above-mentioned hybridoma cell lines C24 and C32 antibodies were determined.

[0070]a. Extraction of RNA: Extract the total RNA of the above-mentioned hybridoma cell lines C24 and C32 with reference to the instructions of the Total Cell RNA Extraction Kit (purchased from Roche Company) and perform reverse transcription immediately;

[0071] 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;

[0072] 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...

Embodiment 3

[0076] Example 3. Recombinant expression and purification of single-chain antibody

[0077] 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 lines C24 and C32, respectively. 3 , introduce six histidines, and optimize the codons of the entire gene according to the preference of the Pichia pastoris expression system to perform recombinant expression of single-chain antibodies. The expressed antibodies were named Antibody A24 and Antibody A32 respectively, and their structures and compositions are shown in the attached figure 2 shown. The recombinant expression of the above-mentioned single-chain antibody has the following characteristics:

[0078] 1. Construction of expression plasmids for fusion protein genes

[0079] The gene sequence of antibody A24 after codon optimization is shown in SEQ ID NO: 19, the amino acid sequence is shown in SEQ ID NO: 17; the ...

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Abstract

The invention relates to an anti-human tissue kallikrein 1 antibody and an application thereof. The invention provides a plurality of monoclonal antibodies. Through matching screening, an antibody combination (A24 and A32) with sensitivity and specificity meeting certain demand is obtained. The antibody combination is convenient for mass production, such that the need of large-scale clinical application in future can be satisfied. The antibody combination is subjected to detection system debugging optimization, such that a human tissue kallikrein 1 enzyme-linked immune quantitative detection kit, a human tissue kallikrein 1 colloidal gold immunochromatographic quantitative detection card and a human tissue kallikrein 1 time-resolved immunofluorescence chromatographic quantitative detection card, which are simple to operate and the sensitivity, selectivity and related detection performances of which can satisfy human plasma sample detection, can be obtained.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry, and in particular relates to an anti-human tissue kallikrein 1 (hK1) antibody and a preparation method thereof and the application of the antibody in the detection of human tissue kallikrein 1. Background technique [0002] Cardiovascular and cerebrovascular diseases are major chronic non-communicable diseases that seriously endanger human health. Among them, coronary heart disease and stroke are the most common causes of death in the world. In my country, with the aging of the population, the incidence, mortality and disability of cardiovascular and cerebrovascular diseases represented by coronary heart disease and stroke are increasing year by year. However, 80% of strokes are preventable. Diabetic nephropathy is the most common complication of diabetes, which seriously endangers the quality of life and medical consumption of diabetic patients. If active intervention measures are not...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40C12N15/13G01N33/573
Inventor 马永赵利利杨芸付红王安良范宇徐春林陈一飞
Owner ZONHON BIOPHARMA INST
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