Human pct fluorescence quantitative detection test card

Abstract

The invention relates to a test paper card for fluorescent quantitative detection of human PCT (procalcitonin). According to the invention, various antibodies are prepared and subjected to pairing screening, and an antibody combination with sensitivity and specificity both meeting requirements is obtained; mass production of the test paper card is facilitated, and the requirement for large-scale clinical application in the future can be met. The antibody combination is subjected to a debugging and optimizing work of a detection system, the time-resolved immunofluorescence chromatography quantitative detection card which is simple and convenient to operate and applied to the human PCT is obtained, and the sensitivity, the specificity and related detection performance of the card can meet the requirements of human clinical 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 with no hormone activity, composed of calcitonin, calcitonin, and N-terminal residue fragments, and is the precursor of calcitonin (CT). The molecular weight is 13KDa, and the half-life is 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...

Examples

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 , the introduction of six histidine tags is shown in SEQ ID NO: 9, the amino acid sequence of the single-chain antibody after the fusion of the histidine tags is shown in SEQ ID NO: 11, and its entire gene is expressed according to the preference of the Pichia pastoris expression system In the method of codon optimization, the nucleotide sequence of the codon-optimized single-chain antibody is shown in SEQ ID NO: 10, and the single-chain antibody is recombinantly expressed. Its structure is as attached figure 2 shown. The recombinant expression steps of the above-mentioned single-chain antibody are as follows:

[0058] a) Expression plasmid construction of single chain antibody gene

[0059] The opt...

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 with no hormone activity, composed of calcitonin, calcitonin, and N-terminal residue fragments, and is the precursor of calcitonin (CT). The molecular weight is 13KDa, and the half-life is 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...

Examples

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 , the introduction of six histidine tags is shown in SEQ ID NO: 9, the amino acid sequence of the single-chain antibody after the fusion of the histidine tags is shown in SEQ ID NO: 11, and its entire gene is expressed according to the preference of the Pichia pastoris expression system In the method of codon optimization, the nucleotide sequence of the codon-optimized single-chain antibody is shown in SEQ ID NO: 10, and the single-chain antibody is recombinantly expressed. Its structure is as attached figure 2 shown. The recombinant expression steps of the above-mentioned single-chain antibody are as follows:

[0058] a) Expression plasmid construction of single chain antibody gene

[0059] The opt...

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