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Reagent kit for detecting concentration of Lp-PLA2 and preparing method thereof

A lipoprotein and phospholipase technology, which is applied to a kit for detecting the concentration of lipoprotein phospholipase A2 in a sample and the field of preparation thereof, can solve the problems of high skill requirements for experimental operators, unfavorable wide-scale promotion of detection, and high detection cost, and achieves Conducive to promotion, easy operation, and low detection cost

Inactive Publication Date: 2015-08-19
北京协和洛克生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the types of reagents commonly used to detect the concentration of Lp-PLA2 on the market are high in detection cost, cumbersome in operation, and require high skills of experimental operators, which is not conducive to the large-scale promotion of this project.

Method used

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  • Reagent kit for detecting concentration of Lp-PLA2 and preparing method thereof
  • Reagent kit for detecting concentration of Lp-PLA2 and preparing method thereof
  • Reagent kit for detecting concentration of Lp-PLA2 and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, preparation of anti-lipoprotein phospholipase A2 mouse monoclonal antibody

[0037] 1 hybridoma cell

[0038] 1.1 Parental cells

[0039] 1.1.1 Myeloma cell lines

[0040] The cell line used for fusion is the Sp2 / 0 cell line routinely used in the preparation of hybridomas. The Sp2 / 0 cells were conventionally cultured in IMDM complete culture medium containing 15% fetal bovine serum under sterile conditions, placed and cultured at 37°C, containing 5% carbon dioxide incubator.

[0041] 1.1.2 Immune spleen cells

[0042] BALB / C mice were immunized with lipoprotein phospholipase A2 pure protein, and the immunization dose was 30mg-60mg / mouse, and the animals were immunized subcutaneously. Blood was collected from the eyeball before immunization, and the serum was separated and stored at -20°C as the serum of normal control mice. In the process of basic immunization, the antigen and complete adjuvant are mixed in equal amounts, and in the booster immunization...

Embodiment 2

[0059] Embodiment 2, the operation and use method of the kit of the present invention are as follows:

[0060] 1. Reconstitute the standard and quality control with deionized water and mix well, respectively add 20ul / well of standard, quality control and sample to the microplate, and 80ul / well of sample buffer, and place it on the shaking reactor React at room temperature for 60 minutes;

[0061] 2. Wash the plate four times with a plate washer and pat dry;

[0062] 3. Add 100ul / well of the enzyme conjugate to the microwell plate, and place it on a shaking reactor for 60min at room temperature;

[0063] 4. Wash the plate four times with a plate washer and pat dry;

[0064] 5. After mixing equal volumes of substrate A and substrate B, add 100ul / well to the microwell plate and react at 37°C for 30min;

[0065] 6. Add stop solution 100ul / well to the microwell plate;

[0066] 7. Use a microplate reader to read the OD value of each reaction well at 450nm;

[0067] 8. Perform q...

Embodiment 3

[0068] Embodiment 3, prepare this kit by the following method:

[0069] Preparation of microwell plates coated with anti-lipoprotein phospholipase A2 monoclonal antibody: Dilute the specific anti-lipoprotein phospholipase A2 monoclonal antibody with a concentration of 6 mg / ml to 10 mM sodium phosphate buffer with a pH value of 7.4 to The concentration is 0.1 μg / ml, add to the concave well of 96-well polystyrene microwell plate, 100ul / well, and coat overnight at 4°C; remove the coating solution, and then wash the microwell plate three times with a plate washer; Then add 10mM sodium phosphate buffer at pH 7.4 containing 1% bovine serum albumin to the wells of the microplate: 300ul / well, seal at room temperature for 6 hours; shake off the blocking solution in the wells, bake at 37°C Dry for 4 hours, seal in a vacuum bag, and store at 4°C;

[0070] Preparation of lipoprotein phospholipase A2 concentration gradient standard and quality control products: use Proclin300 containing 0...

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Abstract

The invention belongs to the technical field of in-vitro immune detection, and particularly relates to a reagent kit for detecting the concentration of Lp-PLA2 and a preparing method of the reagent kit. The reagent kit comprises a platform carrier covered by an Lp-PLA2-resitant specific antibody, an Lp-PLA2 concentration gradient standard product, an Lp-PLA2 quality control product, a horse radish peroxidase labeled Lp-PLA2-resitant antibody, a sample buffer solution, a substrate A, a substrate B, a stop solution and a washing solution. The used detection instruments are simple, and are respectively common use instruments in a laboratory, so that the detection cost is lower, and the popularization is favorably realized. In addition, the operation of the reagent kit is simple, and the goal can be achieved by an ordinary laboratory technicist; meanwhile, the high- specificity and high-affinity Lp-PLA2-resitant specific antibody, the standard product diluent added with unique protein stabilizing agents and a sample buffer solution recipe are adopted in the reagent kit, so that the reliability and the accuracy of the detection results and the stability of the reagent kit are greatly improved.

Description

technical field [0001] The invention belongs to the technical field of in vitro immunoassay, and in particular relates to a kit for detecting the concentration of lipoprotein phospholipase A2 (Lp-PLA2) in a sample and a preparation method thereof. Background technique [0002] Lp-PLA2 belongs to the phospholipase A2 superfamily and is a serine lipase composed of 441 amino acid residues with a relative molecular weight of 45kD. Unlike other members of the phospholipase A2 family, Lp-PLA2 does not require calcium ions to maintain its catalytic activity, and it has the activity of degrading platelet-activating factor, so it is also called platelet-activating factor acetylhydrolase (platelet-activating factor acetylhydrolase, PAF-AH). Lp-PLA2 in the blood is mainly synthesized and secreted by mature macrophages and lymphocytes, exists in large quantities in atherosclerotic sites, and can be regulated by inflammatory mediators. The active Lp-PLA2 can preferentially hydrolyze th...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N33/535
CPCG01N33/573G01N33/535G01N2333/916G01N2800/323
Inventor 郑乐民张立峰李晓燕马志军吴建榕
Owner 北京协和洛克生物技术有限责任公司
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