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C reactive protein detection kit

A technology for detection kits and reactive proteins, applied in the field of molecular immunology, can solve problems such as short half-life

Active Publication Date: 2014-07-23
BEIJING PERGRANDE BIOTECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its half-life is short (4-6 hours), and after active and reasonable treatment, it will quickly return to normal within 3-7 days.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Obtaining of monoclonal antibodies

[0031] 1. Animal immunization

[0032] In order to produce CRP monoclonal antibody, 6-week-old female Balb / c mice weighing about 20 g were selected. For the first immunization, take 20-50 μg of CRP recombinant antigen (purchased from Prospec) and add complete Freund’s adjuvant for subcutaneous multi-point injection. On the 14th and 28th days, the second and third immunization doses are the same as above, plus Freund’s incomplete adjuvant Intraperitoneal injection, booster immunization 3 days before fusion, the appropriate dose is 20-50 μg, and 3 days later, spleen is taken for fusion. Generally, to prepare monoclonal antibodies of murine origin, refer to the method described in the "Antibodies" manual (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp.726, 1988) or Kohler and the technique for preparing hybridomas described by Milstein (Nature, 256:495-497, 1975). ...

Embodiment 2

[0045] Example 2. Preparation of monoclonal antibodies:

[0046] The preserved two strains of hybridoma cells were divided into 1×10 7 The concentration of cells was injected into the peritoneal cavity of BALB / c mice, and the ascites were collected 10 days later. Purify the two monoclonal antibodies separately by the following steps:

[0047] 1. The collected ascites was centrifuged at 2500 rpm, and the supernatant was taken. Add an equal volume of PBS (pH7.4) and 1 / 2 volume of saturated ammonium sulfate, and let stand at 4°C for 30 minutes;

[0048] 2. Centrifuge at 3000rpm, 4°C for 20min;

[0049] 3. Remove precipitation, add an equal volume of saturated ammonium sulfate to the supernatant, and let it stand for 30 minutes;

[0050] 4. Centrifuge at 3000rpm, 4°C for 20min;

[0051] 5. Take the precipitate, add 5ml normal saline and 5ml saturated ammonium sulfate and let stand for 30min;

[0052] 6. Centrifuge at 3000rpm, 4°C for 20min;

[0053] 7. Add 5ml of normal sal...

Embodiment 3

[0060] Embodiment 3. Preparation of detection reagent

[0061] 1. Prepare a multi-well plate coated with CRP monoclonal antibody:

[0062] a) Coating: use 0.05M carbonate buffer solution with a pH value of 9.6-9.8 to mix with an appropriate concentration of a CRP antibody (CGMCC No.8702) collected in Example 2 to prepare a coating mixture, and It was loaded on a microwell plate and coated at 4°C for 12h;

[0063] Carbonate buffer standard formula:

[0064] Anhydrous sodium carbonate 1.600g

[0065] Sodium bicarbonate 2.940g

[0066] Purified water was adjusted to 1000ml.

[0067] b) Plate washing: After diluting 20 times the concentrated washing solution (pH value 6.2-6.6) to 1 times the concentration, wash the plate twice;

[0068] 20 times concentrated lotion standard formula:

[0069]

[0070] c) Blocking: Load the blocking solution (disodium hydrogen phosphate 5.8g, sodium dihydrogen phosphate 0.59g, sodium chloride 8.0g, goat serum 100ml, 0.5ml Proclin-300, purif...

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Abstract

The invention relates to a C reactive protein detection kit, which contains a C reactive protein monoclonal antibody coated porous plate, an enzyme working solution, a sample diluent, an enzyme reaction substrate, a C reactive protein calibrator, and a C reactive protein quality control product. The kit provided by the invention can be used for quantitative detection of the CRP content in serum or plasma to realize early diagnosis of inflammation, and has the advantages of fast detection speed, high sensitivity, and good specificity.

Description

technical field [0001] The invention belongs to the field of molecular immunology, and specifically relates to a C-reactive protein detection kit, a C-reactive protein monoclonal antibody and applications thereof. Background technique [0002] C-reactive protein (CRP) was discovered by Tillett and Francis in 1930 in the serum of patients with pneumonia, a non-specific polysaccharide component of pneumococcus (now called Streptococcus pneumoniae). Substances that precipitate reactions. In 1941, Macleod and Avery successively confirmed that this is a protein that appears during acute infection, and the flocculent precipitation reaction with C polysaccharides depends on the presence of Ca2+. [0003] CRP is non-covalently linked by five identical non-glycosylated subunits, each subunit has a relative molecular mass of 23017 and consists of 206 amino acid residues. The characteristic structure of CRP as a discoid pentamer makes it belong to the family of pentraxins. [0004] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C07K16/18C12N5/20
CPCC07K16/18G01N33/54366G01N33/6893G01N2333/4737
Inventor 于晖李雨心王旭陈勤慧李鑫刘海斌
Owner BEIJING PERGRANDE BIOTECH DEV
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