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Anti-human NMP-22 monoclonal antibody and detection kit thereof

A technology of NMP-22 and monoclonal antibody, applied in the field of molecular immunology, can solve the problems of low sensitivity, time-consuming and labor-intensive kit operation, etc.

Active Publication Date: 2015-11-04
BEIJING PERGRANDE BIOTECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Human NMP-22 detection enzyme immunoassay kit (product number D1100E) manufactured by American Alere company occupies a large proportion in the market of major hospitals and clinical testing institutions, but the market reports that its sensitivity is low, and the operation of the kit is time-consuming and laborious

Method used

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  • Anti-human NMP-22 monoclonal antibody and detection kit thereof
  • Anti-human NMP-22 monoclonal antibody and detection kit thereof
  • Anti-human NMP-22 monoclonal antibody and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Preparation and purification of recombinant human NMP-22 protein

[0051] 1. According to the instruction manual of EASYspin Whole Blood RNA Rapid Extraction Kit (Cat. No. RN06) produced by Beijing Bomed Gene Technology Co., Ltd., extract whole blood RNA.

[0052] 2. According to the instruction manual of the Transcript First-strand cDNA Synthesis SuperMix kit (product number AT301-02) produced by Beijing Quanshijin Biotechnology Co., Ltd., the following reaction system is used to complete the operation:

[0053] cDNA first strand synthesis reaction system

[0054]

[0055] After incubating at 42°C for 30 minutes, reverse transcriptase was inactivated at 85°C for 5 minutes to terminate the reaction.

[0056] Among them, Oligo(DT)18, 2× reaction mixture solution, and enzyme mixture are all from the above kit, and RNA is from the whole blood RNA extracted in step 1.

[0057] 3. Perform PCR reaction to amplify NMP-22 target gene according to the following r...

Embodiment 2

[0071] Example 2. Preparation of anti-human NMP-22 mouse monoclonal antibody

[0072] 1. Select 5 female Balb / c mice aged 6 weeks and weighing about 20 g. The recombinant protein prepared in Example 1 was used for immunization. For the first immunization, take 100 μg of protein and add 0.01M PBS to 500 μl, emulsify with an equal volume of Freund’s complete adjuvant, and inject subcutaneously at multiple points after the emulsification is sufficient. On the 14th and 35th days, the second and third immunization doses were the same as the first immunization, emulsified with Freund's incomplete adjuvant, and then subcutaneously injected at multiple points after emulsification was sufficient. One week after the three immunizations, blood from the tail vein of the mice was collected, and the recombinant antigen was coated with the serum titer by the indirect Elisa method, and the mouse with the highest titer was selected (B mice, see Figure 9 ) spleen for cell fusion. For booste...

Embodiment 3

[0080] Example 3: Preparation of Human NMP-22 Chemiluminescent Kit

[0081] 1. Prepare a microwell plate coated with NMP-22 primary antibody:

[0082] a) Coating: 0.05M carbonate buffer solution with a pH value of 9.6 and the mouse monoclonal antibody with the clone number 5E8 prepared in Example 2 at an appropriate concentration were used to prepare coating solutions with different concentrations. Select the coating concentration of 5 μg / ml, 2.5 μg / ml, 1.25 μg / ml, 0.625 μg / ml, and load the coating solution on the microplate at 120 μl / well, and coat at 4°C for 12 hours;

[0083] Carbonate buffer standard formula:

[0084]

[0085] b) Washing plate: dilute 20 times concentrated lotion to 1 times concentration, wash the plate twice with 1 times concentration lotion; standard formula of 20 times concentrated lotion:

[0086]

[0087] c) Blocking: Load the blocking solution (disodium hydrogen phosphate 5.8g, sodium dihydrogen phosphate 0.593g, sodium chloride 8.0g, goat se...

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Abstract

The present invention relates to an anti-human NMP-22 monoclonal antibody and a detection kit thereof, and particularly provides human nuclear matrix protein 22 (NMP-22) that is expressed by using a gene cloning recombinant technology, and an anti-human NMP-22 mouse monoclonal antibody is obtained by application of an immunological technology. The present invention further discloses a kit for specific detection of NMP-22 in a human sample. The kit comprises two specific anti-human NMP-22 monoclonal antibodies. The kit disclosed by the invention can be used for carrying out quantitative detection on the content of NMP-22 in a sample quickly, sensitively and specifically.

Description

technical field [0001] The disclosure belongs to the field of molecular immunology, and specifically relates to recombinantly expressed human nuclear matrix protein 22 (NMP-22) and its preparation and purification methods; monoclonal antibodies against human NMP-22 obtained by applying immunological techniques; and human NMP-22 22 detection kits. Background technique [0002] Bladder tumors are the most common tumors of the urinary system, and also the most common tumors of the urinary system in my country, of which more than 90% are bladder transitional cell carcinomas (bladder transitional cell carcinoma, BTCC). [0003] The biological behavior of BTCC is complex and changeable, and the salient features are high recurrence rate and low malignancy of tumor biological behavior. BTCC is prone to recurrence, multiple, infiltration and metastasis. Therefore, these factors are likely to affect the diagnosis and treatment of bladder cancer. Early diagnosis is relatively limite...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K14/47C12N15/12G01N33/574
Inventor 于晖王旭陈勤慧李雨心詹春光
Owner BEIJING PERGRANDE BIOTECH DEV
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