Early stage predetection acute coronary comprehensive diagnosis kit

A technology for diagnosis kits and syndromes, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems that ordinary ELISA kits cannot meet the detection requirements, and achieve the effects of improving specificity, improving sensitivity, and simplifying experimental procedures

Inactive Publication Date: 2007-01-31
GUANGZHOU FIRST PEOPLES HOSPITAL (GUANGZHOU DIGESTIVE DISEASE CENT GUANGZHOU FIRST PEOPLES HOSPITAL GUANGZHOU MEDICAL UNIV THE SECOND AFFILIATED HOSPITAL OF SOUTH CHINA UNIV OF TECH)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because its serum content is at the level of "μg / L", ordinary ELISA kits can no longer meet the detection requirements, so there is an urgent need to develop an ultra-sensitive ELISA kit for detecting sCD40L, so as to greatly improve the clinical treatment effect and improve the cure rate of patients

Method used

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  • Early stage predetection acute coronary comprehensive diagnosis kit
  • Early stage predetection acute coronary comprehensive diagnosis kit
  • Early stage predetection acute coronary comprehensive diagnosis kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1, cloning, expression, purification, and identification of sCD40L:

[0030] The human sCD40L gene was searched from Gene Bank, and primers were designed. After RT-PCR amplification, the cDNA was cloned into pGEM-T vector and confirmed by sequencing. Then subclone into the pET-28a prokaryotic expression vector with N-broken His tag, extract the plasmid, and double-enzyme-digest identification; identify the correct recombinant to transform BL21 competent cells, extract the plasmid, and double-enzyme-digest identification; pick the colony containing the recombinant , shake culture at 37°C, induce the expression of the target protein with IPTG; detect the expression of the target protein by SDS-PAGE electrophoresis, optimize the expression conditions, so that the target protein can be expressed in a soluble form; and column purification of the target protein with His tag, SDS-PAGE separation and identification of the expressed target protein.

Embodiment 2

[0031] Example 2, preparation, purification, identification, and labeling of anti-sCD40L monoclonal antibodies:

[0032] Prokaryotic expression of purified His-sCD40L protein, immunization of BALB / c mice at intervals of 3 weeks and booster immunization twice, 2 weeks after the last booster immunization, splenocytes were taken to fuse with SP2 cells, HAT selection medium, cultured for 3 weeks, Take the supernatant and use the purified His-sCD40L to initially screen the monoclonal antibody, and then use the sCD40L protein-coated ELISA plate to detect the antibody, pick the antibody-positive cell lines, monoclonize them, and culture them in HT selection medium for three weeks. The supernatant was tested for antibodies by ELSIA, the positive clones were expanded and cultured, and the cell lines were injected into the peritoneal cavity of BALB / c mice. After 2 weeks, ascitic fluid was collected, aliquoted, and stored at -20°C. The prepared monoclonal antibodies were verified by West...

Embodiment 3

[0033] Example 3, preparation, purification, identification of anti-sCD40L polyclonal antibody:

[0034] Rabbits were immunized with purified sCD40L protein, immunized twice, with an interval of 1 month; 4 ) 2 SO 4 The IgG was preliminarily purified from rabbit serum by precipitation method, and then the anti-sCD40L IgG was further purified by self-made sCD40L-Sepharose affinity chromatography column, and the polyclonal antibody was identified by SDS-PAGE and Western Blot.

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Abstract

The invention discloses a diagnose kit for early stage forcast ACS. It is made up form sCD40L standard albumen used as biochemical criterion, single clone antibody and multi-clone antibody including sCD40L, sCD40L single antibody marked by biotin as second antibody. It uses multi sCD40L clone antibody to cover enzyme scale, uses HRP-poly-lysine as signal testing amplification system. The diagnose kit is a super sensitive kit for testing serum sCD40L ELISA, and could improve curative ratio.

Description

technical field [0001] The invention relates to a diagnostic kit for early prediction of acute coronary syndrome (ACS), in particular to preparation of monoclonal and polyclonal sCD40L antibodies using prokaryotic expression of human soluble CD40 ligand (sCD40L) protein as an antigen; HRP- Polylysine-streptavidin complex as a detection signal amplification system to detect serum sCD40L ultrasensitive ELISA kit for the risk of cardiovascular events in ACS [0002] technology field. Background technique [0003] ACS is a common and frequently-occurring disease that seriously threatens human life and health. Therefore, early prediction of the risk of this disease is of great significance for effective prevention and treatment measures in clinical practice. However, the current clinical control of the disease is not ideal, mainly because the early onset risk prediction of ACS cannot be made, thus delaying the treatment of the disease. To diagnose ACS, the main biochemical exam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/543G01N33/531
Inventor 徐邦牢贝春花王蓉刘万里张革
Owner GUANGZHOU FIRST PEOPLES HOSPITAL (GUANGZHOU DIGESTIVE DISEASE CENT GUANGZHOU FIRST PEOPLES HOSPITAL GUANGZHOU MEDICAL UNIV THE SECOND AFFILIATED HOSPITAL OF SOUTH CHINA UNIV OF TECH)
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