Monoclonal antibody specifically combined with HFABP (heart fatty acid binding protein) and applications thereof

A monoclonal antibody and protein technology, applied in anti-animal/human immunoglobulin, microbial-based methods, biochemical equipment and methods, etc.

Inactive Publication Date: 2015-06-24
HUNAN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no high-quality supply of raw materials for HFABP testing in China, and the key raw materials for testing are completely dependent on imports, but imported antibodies are expensive, which brings a heavy economic burden to patients

Method used

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  • Monoclonal antibody specifically combined with HFABP (heart fatty acid binding protein) and applications thereof
  • Monoclonal antibody specifically combined with HFABP (heart fatty acid binding protein) and applications thereof
  • Monoclonal antibody specifically combined with HFABP (heart fatty acid binding protein) and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1. Expression and Purification of Recombinant Strain HFABP Protein

[0043] The recombinant prokaryotic expression plasmid of HFABP was transformed into Escherichia coli ER2566, and spread to the + After culturing at 37 °C for 12 h, a single colony was picked from the plate and inoculated into 10 mL LB medium (containing Kan + ), cultured at 37 ℃ for 8 h, and then inoculated 10 mL of cultured bacteria into 1L LB medium (containing Kan + ) cultured in the bacterial solution OD 600 When the value reached 0.6, the expression was induced by adding IPTG at a final concentration of 0.4 mmol / L. After 4 hours, collect the cells by centrifugation at 12,000 g at room temperature for 5 minutes, add 40 mL of cell lysate (50 mmol / L Tris HCl, 1 mmol / L EDTA, 100 mmol / L NaCl, pH 8.0), Ф10 mm probe with 200W power The cells were sonicated, centrifuged at 12,000 g for 10 min at 4°C, and the supernatant was collected. The supernatant was precipitated with ammonium sulfate, dialyzed...

Embodiment 2

[0049] Freund's complete adjuvant and Freund's incomplete adjuvant containing heat-inactivated Mycobacterium tuberculosis were shaken and mixed thoroughly on a vortex shaker to ensure that the bacterial solution was fully suspended. Phosphate buffer and 1.0 mg / mL recombinant HFABP were mixed at 1:1 (V / V), and then the mixture was pumped repeatedly with a 1.0 mL syringe until the recombinant protein and adjuvant were fully mixed to form a homogeneous emulsion.

[0050] Inject 6-8 w SPF female BALB / c mice with complete Freund's adjuvant and heat-inactivated Mycobacterium tuberculosis emulsified immunization antigen HFABP to each mouse with 40 μg of antigen from the hock joint, the first immunization Two weeks later, each mouse was re-injected with 40 μg antigen from the hock joint with incomplete Freund's adjuvant and heat-inactivated Mycobacterium tuberculosis emulsified immunization antigen HFABP. Eight days after immunization, blood was collected from the tail vein of the mic...

Embodiment 3

[0055] 1. Select sp2 / 0 myeloma cells in good growth state and lymphocytes from immunized mice to mix at a ratio of about 1:2-1:3, centrifuge at 1200 g for 2 min, and discard the supernatant. Tap the centrifuge tube to disperse the cells evenly, slowly add 1 mL of 37 ℃ preheated PEG 1500, mix gently, and immediately add 20 mL of serum-free 1640 medium after 60 s. After centrifugation at 1000 g for 2 min, discard the supernatant, add 20% FBS serum HAT-1640 medium to the cell pellet and mix gently, evenly spread in 96-well plate, 200 μL per well, at 37 °C in 5% CO 2 cultured in an incubator. After 5-7 days, observe the fusion effect and change the medium. After 3 days, take the supernatant and use the indirect ELISA method to screen positive clones. Select wells with a higher ratio of positive value to cell number, and perform subcloning by limiting dilution method. After 4 times of subcloning, a hybridoma cell line capable of stably secreting antibodies is obtained.

[0056]...

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Abstract

The invention discloses a hybridoma cell strain which can stably secrete a high-titer anti-human HFABP (heart fatty acid binding protein). The Preservation No. of the hybridoma cell strain is CCTCC NO: C2014196; and by taking a purified recombinant expressed HFABP as an antigen for immunizing a female BALB / c mouse, lymphocytes of the immunized mouse are fused with sp2 / 0 cells, and through indirect ELISA (enzyme-linked immuno sorbent assay) and Western blotting detection, the titers respectively reach 1:104 and 1:4000. A monoclonal antibody secreted by the cell strain has good specificity and relative affinity, can be used as a key raw material for the early detection of HFABP, and provides an important raw material for the establishment of a clinical diagnostic reagent which is applied to the rapid and high-sensitivity detection of HFABP and comprises an immune-gold label, an immune test strip, an enzyme-linked immunoreagent, immunoturbidimetry, and the like; and the monoclonal antibody can also be used as an important raw material of a diagnostic reagent which is used for detecting the HFABP of the cerebrospinal fluid, and monitoring and predicting the transformation, from mild cognitive impairment to paraphrenia, of old people.

Description

technical field [0001] The invention relates to a specific monoclonal antibody against human heart fatty acid binding protein, which can be used for early diagnosis, risk assessment and prognosis judgment of myocardial injury. Background technique [0002] Cardiovascular disease has become the number one killer of human health in the 21st century, the most dangerous of which is Acute Coronary Syndrome (ACS), which includes unstable angina and acute myocardial infarction (AMI). Statistics show that 17 million people die from cardiovascular diseases every year in the world, and more than half of them die from acute myocardial infarction. In the past ten years, the incidence of acute myocardial infarction in my country has been showing an obvious upward trend, which is close to the international average level. The onset of acute myocardial infarction is sudden, and the mortality rate in the acute phase is about 30%. The main cause of acute myocardial infarction is the sudden ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/68G01N33/577C12R1/91
Inventor 刘如石邱义兰廖旻晶邱果
Owner HUNAN NORMAL UNIVERSITY
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