Bovine derived creatine kinase isoenzyme double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) rapid detection kit

A double-antibody sandwich and creatine kinase technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of secondary infection of implanted pathogenic bacteria, low early diagnosis rate, etc., achieve short detection time, simple operation, Simple operation effect

Active Publication Date: 2017-09-05
INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the diagnosis of dairy cow endometritis mainly relies on the traditional methods of inspection, palpation and uterine probe, which has the problems of low early diagnosis rate and secondary infection of implanted pathogenic bacteria

Method used

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  • Bovine derived creatine kinase isoenzyme double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) rapid detection kit
  • Bovine derived creatine kinase isoenzyme double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) rapid detection kit
  • Bovine derived creatine kinase isoenzyme double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) rapid detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Correlation between creatine kinase in dairy cow serum and endometritis

[0032] Obtain the serum of cattle diagnosed with endometritis by conventional methods, detect the creatine kinase (CK) value, and determine the correlation between bovine endometritis and CK by comparing the control group with the positive group.

[0033] A number of serums from cows with clinical symptoms of endometritis were collected from cattle farms in Hohhot and Baotou. Biochemical analysis was performed to detect the activity of creatine kinase and relevant data were obtained. Using bovine heart-derived creatine kinase (CK-MB) as a standard, SDS-PAGE electrophoresis was used to detect bovine serum samples with endometritis (Figure 1), and the clinical serum sample CK and bovine heart origin were judged by observing the size and color of the target band. Creatine kinase (CK-BB) was used as the consistency and compliance of the standard. Preliminary research results are as follows...

Embodiment 2

[0038] Embodiment 2. Whole gene synthesis bovine creatine kinase isoenzyme CK-BB

[0039] Since bovine endometritis is highly correlated with bovine creatine kinase isoenzyme CK-BB in serum, bovine creatine kinase isoenzyme CK-BB was used as antigen to prepare related monoclonal and polyclonal antibodies. In the present invention, the shortage of creatine kinase CK-BB, a key reagent derived from bovine brain or smooth muscle, severely restricts the development of the experiment. Due to the small application of CK-BB, most of the bovine creatine kinases on the market have CK-MB as the main component, and there are no commercial products of CK-BB, which has caused difficulties for research.

[0040] In order to solve the problem of insufficient CK-BB, the inventor decided to use the method of total gene synthesis to synthesize the bovine CK-BB gene and perform prokaryotic expression on it. The amino acid sequence of bovine creatine kinase CK-BB was queried through NCBI, the seq...

Embodiment 3

[0058] Example 3. Preparation of CK-BB monoclonal antibody

[0059] 1. Small test: The PET28b-CK-BB positive plasmid was transformed into BL21 and Rosetta host bacteria, and the expression was obvious in the small test.

[0060] 2. Antigen preparation: A large amount of PET28b-CK-BB (BL21) was induced to express, the target protein was clearly expressed in the supernatant and inclusion bodies, the supernatant was purified first, and the supernatant was purified and dialyzed to finally obtain 5 mg of CK-BB recombinant protein with a purity of >90%.

[0061] 3. Preparation of polyclonal antibodies: Polyclonal antibodies were prepared with a titer of 1:243,000.

[0062] 4. Preparation of monoclonal antibody: Monoclonal antibody was prepared, and 5 positive cell lines were screened.

[0063] 5. Antibody preparation and purification: Ascites was planted and purified to obtain monoclonal antibodies.

[0064] 6. Antibody pairing screening: ELISA method is used for pairing screenin...

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Abstract

The invention relates to a bovine derived creatine kinase isoenzyme (CK-BB) double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) rapid detection kit and bovine derived creatine kinase isoenzyme (CK-BB) detection method. The rapid detection kit has the advantages of quantitative property, wide detection concentration range, high sensitivity, strong specificity, good repeatability, short detection time and the like. A novel early and rapid cow endometritis diagnosis method is built, and rapid detection of cow endometritis and auxiliary clinic examination diagnosis are realized.

Description

technical field [0001] The invention relates to the field of rapid detection of colloidal gold immunochromatography, in particular to a rapid detection kit and method for double-antibody sandwich ELISA of bovine creatine kinase isozyme (CK-BB). Background technique [0002] In recent years, my country's dairy industry has developed very rapidly, but infertility has an important impact on the development of dairy industry. A large number of data show that infertility accounts for 20% to 30% of dairy cow diseases, and endometritis accounts for 40% to 60% of infertility. There are many causes of cow endometritis, but the main reason is the early postpartum infection. Studies have shown that 93% of cows have uterine infection bacteria in the early postpartum stage, and the infection rate is reduced to 39% by 46-60 days. %. [0003] Endometritis in dairy cows prevents fertilized eggs from implanting or early death of embryos, delays the calving interval, seriously affects the r...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/573
CPCG01N33/573G01N33/577
Inventor 赵世华杨鼎达来宝力格刘威王根云兰儒冰高娃杨斌张月梅宋越张帆戴伶俐陈伟宋爱军
Owner INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
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