Human Tissue Kallikrein 1 Elisa Quantitative Detection Kit

Abstract

The invention relates to an ELISA quantitative detection kit for detecting human tissue kallikrein 1 (hK1) and related antibodies. According to the invention, a plurality of monoclonal antibodies are prepared and are paired and screened, so an antibody combination (A24 and A32) with sensitivity and specificity meeting demands is obtained; and the antibody combination can be conveniently produced in a large scale and can meet demands of large-scale clinical application in the future. The antibody combination is subjected to adjustment and optimization of a detection system so as to obtain the ELISA quantitative detection kit for human tissue kallikrein 1 with the advantages of simple operation and good sensitivity, specificity and related detection performance.

Description

technical field

[0001] The invention belongs to the technical field of immunochemistry, and in particular relates to an ELISA quantitative detection kit for detecting human tissue kallikrein 1 (hK1) and related antibodies. 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 taken from the perspective of evidence-based medicine, dia...

Examples

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 ...

technical field

[0001] The invention belongs to the technical field of immunochemistry, and in particular relates to an ELISA quantitative detection kit for detecting human tissue kallikrein 1 (hK1) and related antibodies. 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 taken from the perspective of evidence-based medicine, dia...

Examples

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 ...

Images