Method of determining ceruloplasm concentration in blood cake and hepatolenticular degeneration disease screening reagent box and diagnostic reagent

A technology of ceruloplasmin concentration and concentration, which is applied in the directions of disease diagnosis, biological testing, biochemical equipment and methods, etc., can solve the problems of lack of ceruloplasmin, difficult to measure ceruloplasmin, and difficult to use.

Inactive Publication Date: 2002-11-13
CHI NAK BEK
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0008] The method for the determination of ceruloplasmin oxidase activity has no specific enzyme substrate for ceruloplasmin, so it is difficult to measure ceruloplasmin
In the measurement method using polyclonal antibodies, polyclonal antibodies bind to both holoceruloplasmin and apoceruloplasmin, so they have low specificity
[0009] Moreover, in order to achieve the above method, a large amount of blood must be collected, and the serum should be separated through an additional blood centrifugation process. After collection, due to the stability of the protein in the serum, it is necessary to keep the frozen state at important moments in the process of preservation and handling, which has great potential inconvenience
As mentioned above, the existing method is inconvenient in terms of collection, transportation, storage, etc., and the number of samples that can be measured at one time is also limited. Therefore, this method is not easy to use in national screening inspections that measure a large number of samples

Method used

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  • Method of determining ceruloplasm concentration in blood cake and hepatolenticular degeneration disease screening reagent box and diagnostic reagent
  • Method of determining ceruloplasm concentration in blood cake and hepatolenticular degeneration disease screening reagent box and diagnostic reagent
  • Method of determining ceruloplasm concentration in blood cake and hepatolenticular degeneration disease screening reagent box and diagnostic reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Production of ceruloplasmin-specific polyclonal antibody

[0046] Purified human ceruloplasmin containing whole ceruloplasmin purchased from Sigma was used in this example. In order to generate a polyclonal antibody specific to an antigen, 400 μl of a refined ceruloplasmin solution dissolved in a phosphate buffer was emulsified with Freund's adjuvant (marketed by BRL Company) at a concentration of 1 mg / ml, and given to 10-week-old rabbits received 3 intradermal injections at 14-day intervals. After 14 days, 400 μl of refined ceruloplasmin solution dissolved in phosphate buffer was injected into the ear vein at a concentration of 1 mg / ml, and blood was collected 7 days later by perforating the heart. Place the collected blood at room temperature for 30 minutes, and place it overnight at 4°C. After complete coagulation, centrifuge at 2,500 rpm for 30 minutes, and take supernatant and serum. Ammonium sulfate was added to the collected serum so that the final ...

Embodiment 2

[0047] Example 2: Production of ceruloplasmin-specific monoclonal antibody

[0048] The monoclonal antibody used in the present invention is 100 μl of purified ceruloplasmin at a concentration of 1 mg / ml, vulcanized with the same amount of Freund's adjuvant, and injected 3 times at intervals of 2 weeks after 6-8 weeks. BALB / c mice intraperitoneally. After the final injection, the production of anti-ceruloplasmin antibody was confirmed, and 2 weeks later, the final immunization was performed with 100 μg of ceruloplasmin. After 3 days, spleen cells were extracted from the mice, mixed with Sp2 / 0-Ag14 myeloma cells at a ratio of 10:1, and the mixture was placed in 50% polyethylene glycol 1500 solution for 3 minutes to carry out cell fusion. Then centrifuge this at 1,200rpm for 8 minutes to obtain the cell pellet, suspend it in HAT RPMI-1640 culture medium containing 10% fetal bovine serum, so that each ml contains 3.5×10 6 Cell. Afterwards, dispense into 96-well plates, 0.1ml p...

Embodiment 3

[0050] Example 3: Confirmation of neutralization of ceruloplasmin oxidase activity by antibodies

[0051] After mixing 10 μg of purified ceruloplasmin with the hybridoma culture supernatant of the same volume obtained in Example 2, they were reacted at a temperature of 37° C. for 30 minutes, and then, at a temperature of 4° C. ) acrylamide gel (7.5%) electrophoresis. After electrophoresis, in order to confirm the activity of ceruloplasmin oxidase, a sodium acetate (0.1 M, pH 5.7) buffer solution having a concentration of 1 mg / ml p-phenylenediamine was used as a staining solution, and the gel was stained at a temperature of 37°C. Colloid (gel) for 2 hours, decolorized in 50% ethanol solution. ( figure 1 )

[0052] From figure 1 It can be seen from the results that purified ceruloplasmin oxidizes colorless p-phenylenediamine due to its own oxidase activity, forming a purple band. In contrast, in the mixture of ceruloplasmin and ceruloplasmin-specific antibody, no purple ban...

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Abstract

The present invention relates to a method for measuring the concentration of whole plasma ceruloplasmin, in particular to the use of whole plasma ceruloplasmin-specific polyclonal antibody and whole plasma ceruloplasmin-specific monoclonal antibody, by using enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay method) Immunosorbent assay; ELISA) or dissociation-enhanced time-resolved fluorescent immunoassay (dissociation-enhanced time-resolved fluoroimmunoassy) to obtain the standard concentration curve, the method of measuring the concentration of whole plasma ceruloplasmin.

Description

technical field [0001] The present invention relates to a method for measuring the concentration of holoceruloplasmin (holoceruloplasmin), in particular to the use of holoceruloplasmin-specific polyclonal antibodies and holoceruloplasmin-specific monoclonal antibodies, by enzyme-linked immunosorbent assay ( The standard concentration curve obtained by enzyme-linked immunosorbent assay (ELISA) or dissociation-enhanced time-resolved fluoroimmunoassy (dissociation-enhanced time-resolved fluoroimmunoassy) is a method for determining the concentration of ceruloplasmin in whole plasma. Background technique [0002] The method of measuring the concentration of whole plasma ceruloplasmin plays a very important role in the diagnosis of Wilson's disease. [0003] Wilson's disease, also known as hepatolenticular degeneration (Wilson's disease), was first reported in 1912. It is an autosomal recessive disease that occurs at a frequency of 1 in 25,000-30,000 people in the world today. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/68
CPCG01N33/6893G01N2800/02Y10S435/975G01N33/577
Inventor 韩始薰章暎珠李秀英申夏澈朴善荣柳恩善韩熙成
Owner CHI NAK BEK
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