Monoclonal antibody against human lp-pla2 protein resistant to lipoprotein interference and its application

A monoclonal antibody and protein technology, applied in the direction of antibodies, immunoglobulins, antibody medical components, etc., can solve the problem of increasing the difficulty of developing monoclonal antibodies for diagnosis, the inability to produce monoclonal antibodies against Lp-PLA2, and the inability to accurately identify them, etc. problems, to achieve good specificity and temperature stability, good affinity and specificity, and good specificity

Active Publication Date: 2018-02-23
VAZYME BIOTECH NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no domestic production of monoclonal antibodies against Lp-PLA2 that can be used in clinical diagnosis
Most of Lp-PLA2 binds to lipoproteins in blood, and the epitopes targeted by Lp-PLA2 monoclonal antibodies produced based on recombinant antigens are likely to be covered by lipoproteins, making these monoclonal antibodies unable to accurately identify natural Lp-PLA2 in blood Lp-PLA2 protein, which increases the difficulty of developing monoclonal antibodies for diagnostic use

Method used

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  • Monoclonal antibody against human lp-pla2 protein resistant to lipoprotein interference and its application
  • Monoclonal antibody against human lp-pla2 protein resistant to lipoprotein interference and its application
  • Monoclonal antibody against human lp-pla2 protein resistant to lipoprotein interference and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Expression Purification and Purity Identification of Recombinant Lp-PLA2 Protein

[0050] The prokaryotic expression plasmid containing the Lp-PLA2 gene sequence was transformed into Escherichia coli BL21(DE3), and cultured on LB plate. Pick a single colony from the plate and inoculate it into LB medium. After expanding the culture, when the OD600 value of the bacterial solution reaches 0.6-0.8, add IPTG with a final concentration of 0.2mM to induce expression. After 16 hours, the bacterial cells were collected by centrifugation, and the supernatant was collected by centrifugation after the bacteria were disrupted by ultrasonic. The supernatant was purified by cation exchange column and molecular sieve chromatography column, and the samples were collected and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for protein purity.

[0051] Carry out sodium dodecylsulfonate-polyacrylamide gel electrophoresis after purifying recombinant Lp-PL...

Embodiment 2

[0052] Example 2 Animal immunity and serum titer detection

[0053] Mix and emulsify the purified recombinant Lp-PLA2 protein with a concentration of 1 mg / ml and the same amount of complete Freund's adjuvant with double syringes, and immunize with the emulsified antigen for 6-8 weeks. Specific pathogen-free female BALB / c For mice, each mouse was injected with 40 μg antigen from the hock joint. After 2 days, the purified recombinant Lp-PLA2 protein with a concentration of 1 mg / ml and the same amount of Freund's incomplete adjuvant were mixed and emulsified with double syringes, and each mouse was injected with 40 μg emulsified Lp-PLA2 again from the hock joint. antigen. Eight days later, blood was collected from the tail vein, centrifuged to obtain the supernatant, and the serum titer was detected by indirect ELISA. After the second immunization, the titer of the serum after a million-fold dilution was as high as 2.0 or more (Table 1). Select the serum titer greater than 10 ...

Embodiment 3

[0056] Example 3 Cell Fusion, Positive Hybridoma Screening and Subcloning

[0057] 1. Preparation and screening of high-titer hybridoma cell lines

[0058] Using PEG 1500 as a fusion agent, sp2 / 0 myeloma cells and lymphocytes from immunized mice were fused, and the ratio of the two cells was 1:2-1:3. The fused cells were cultured in HAT-1640 medium containing 20% ​​FBS serum. One week later, observe the fusion state and change the medium, and take the cell culture supernatant 3 days after the medium change. The ELISA plate was coated with the serum of patients with clinically confirmed atherosclerosis, which contains a high concentration of natural Lp-PLA2 protein, and the positive clones were screened by indirect ELISA method, and the cells with a higher ratio of positive value to cell number were selected for multiplication. After subcloning, multiple strains of hybridoma cell banks capable of stably secreting monoclonal antibodies against Lp-PLA2 protein were finally obta...

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Abstract

The invention discloses a monoclonal antibody of antihuman Lp-PLA2 protein for resisting lipoprotein interference and application thereof. The invention discloses a hybridoma cell strain LP1 capable of stably secreting the monoclonal antibody of antihuman lipoprotein related phospholipase A2 protein for resisting lipoprotein interference, the conservation number is CCTCC NO:C2016204. Through the detection, the affinity of the monoclonal antibody achieves 1*10<-12>M, and the affinity cannot be influenced by the combination of the lipoprotein and the Lp-PLA2 protein. The antibody has good stability and can be used as the key raw material for preparing the Lp-PLA2 diagnostic reagent. A diagnostic detection kit with good specificity and high accuracy can be developed by use of the monoclonal antibody, and the difference of the Lp-PLA2 in the serum of healthy human and the patient with the cardiovascular disease can be successfully detected.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a lipoprotein interference-resistant anti-human Lp-PLA2 protein monoclonal antibody and application thereof. Background technique [0002] Atherosclerosis (AS) is the main cause of coronary heart disease, stroke and peripheral vascular disease. Lipid metabolism disorder and its accompanying vascular inflammation are the basis of atherosclerosis, which is characterized by the involvement of arterial lesions starting from the intima, usually with accumulation of lipids and complex carbohydrates, hemorrhage and thrombosis, and then fibrous tissue Hyperplasia and calcium deposition, and gradual degeneration and calcification of the arterial media, resulting in thickening and hardening of the arterial wall and stenosis of the vascular lumen. Lesions often involve large and medium muscular arteries, and once they develop enough to block the lumen of the artery, the tiss...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/40G01N33/577G01N33/573A61K39/395A61P9/00
CPCA61K39/00C07K16/40C07K2317/33C07K2317/92C07K2317/94
Inventor 曹林唐波牛英波徐晓昱杨晨辰
Owner VAZYME BIOTECH NANJING
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