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Human apolipoprotein B100 (ApoB100) monoclonal antibody and chemiluminescence immune assay determination kit adopting the human ApoB100 monoclonal antibody

A monoclonal antibody, apolipoprotein technology, applied in the biological field, can solve the problems of long-term storage, false positives, false negatives, etc., and achieve the effects of improved sensitivity, low production cost, and wide detection range

Active Publication Date: 2014-12-03
大庆麦伯康生物技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Apo B currently tested in clinical laboratories 100 The most common methods are radioimmunoassay (RIA), immunoturbidimetry and enzyme-linked immunosorbent assay (ELISA), in which RIA must be labeled with radioactive elements, the detection equipment is complex and expensive, and the half-life of radioactive elements is short and cannot be preserved for a long time. The results are unstable, and the radioactive contamination also brings harm to the experiment operators; the enzyme immunoassay method has low sensitivity and many influencing factors, which can easily cause false negatives and false positives

Method used

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  • Human apolipoprotein B100 (ApoB100) monoclonal antibody and chemiluminescence immune assay determination kit adopting the human ApoB100 monoclonal antibody
  • Human apolipoprotein B100 (ApoB100) monoclonal antibody and chemiluminescence immune assay determination kit adopting the human ApoB100 monoclonal antibody
  • Human apolipoprotein B100 (ApoB100) monoclonal antibody and chemiluminescence immune assay determination kit adopting the human ApoB100 monoclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1: the preparation of hybridoma

[0041] 1. Immunization of mice: The immunized animals were 8-week-old male Balb / C mice, mixed with human ApoB100 with a protein content of 50ug and complete Freund's adjuvant, and after fully emulsified, take multiple points on the back, and underarms, Inguinal immunization; 15 days later, the same dose was mixed with Freund's incomplete adjuvant for the second time; 15 days later, the same dose was mixed with Freund's complete adjuvant for the third time; 10 days later, blood was collected from the tail and measured by indirect ELISA method For serum titer, boost immunization with the same dose of pure antigen without adjuvant; take splenocytes for fusion after 3 days;

[0042] 2. Cell fusion: Sp2 / 0 cells in the logarithmic growth phase were mixed with splenocytes at a ratio of 1:10, and hybridoma cells were obtained by the polyethylene glycol (PEG) method, named 4-3-2 and 4-6 -3. The obtained hybridoma cells were suspende...

Embodiment 2

[0043] Example 2: Screening of Monoclonal Antibodies

[0044] The supernatant of the culture medium was recovered from the wells in which the hybridoma cells obtained in Example 1 were cultured, and the monoclonal antibody reacting with the antigen polypeptide in the ELISA method was selected.

[0045] First, 100uL of human ApoB at a concentration of 0.2ug / ml 100 Put them into each well of a 96-well plate, fix them on the solid phase after overnight at 4°C, and then block with 150uL bovine serum albumin at a concentration of 1% for 2 hours. Add 100uL hybridoma culture medium supernatant to each well, react at 37°C for 2 hours, then add horseradish peroxidase-conjugated anti-mouse antibody diluted 10,000 times and react at 37°C 1 hour. Color development was performed using tetramethylbenzidine microperoxidase substrate (TMB) as substrate. After adding 50 uL of 0.2N sulfuric acid to terminate the reaction, measure the absorbance at 450, select 2C6 and 3E8 with absorbance abou...

Embodiment 3

[0048] Example 3: Labeling of Monoclonal Antibodies

[0049] Monoclonal antibody 4-6-3 was used for HRP labeling according to conventional methods. The titer of the labeled monoclonal antibody was determined by the following method. Human ApoB at a concentration of 0.2ug / ml 100 Immobilized on a 96-well microplate (100uL / well). Use 1% bovine serum albumin to block for 2 hours, add labeled monoclonal antibody (diluted 100 times from the first well), make 4-fold dilution from the second well, and react at room temperature for 2 hours. After addition of TMB, the reaction was carried out at room temperature for 20 minutes and quenched with 0.2N sulfuric acid. The absorbance at 450 nm was measured, and the titer against the antigen immobilized in the solid phase was obtained in the manner in Example 2. The results show that there is an effective titer ( figure 2 ).

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Abstract

The invention provides a chemiluminescence immune assay determination kit adopting a human apolipoprotein B100 (ApoB100) monoclonal antibody and a preparation method thereof. The invention also provides the human ApoB100 monoclonal antibody which can specifically recognize human ApoB100, a hybrid tumor for producing the human ApoB100 monoclonal antibody, and a method for high-sensitivity detection of human ApoB100 by the human ApoB100 monoclonal antibody. The chemiluminescence immune assay determination kit has high sensitivity, a wide detection scope and a low cost and is convenient for operation.

Description

【Technical field】 [0001] The present invention relates to the identification of human apolipoprotein B 100 (Apo B 100 ), a hybridoma producing the monoclonal antibody, a Chemiluminescent Immunoassay Quantitative Assay (CLIA) kit and a preparation method thereof, the invention belongs to the field of biotechnology. 【Background technique】 [0002] Cardiovascular and cerebrovascular disease is a common disease that seriously threatens the health of human beings, especially middle-aged and elderly people over 50 years old. The number of people who die from cardiovascular and cerebrovascular diseases in the world is as high as 15 million every year, ranking first among various causes of death. Cardiovascular and cerebrovascular diseases have the characteristics of "high morbidity, high disability rate, high mortality, high recurrence rate, and many complications", that is, "four highs and one high". At present, there are more than 270 million patients with cardiovascular and cer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/68G01N33/545C12R1/91
Inventor 蓝兴国李玉花张晓磊李春雷冯玥
Owner 大庆麦伯康生物技术有限公司
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