Double-antibody sandwich enzyme-linked immunosorbent assay kit for detection of Streptococcus agalactiae

An immunoadsorption and streptococcus lactis technology, which is applied in the field of detection and immunodiagnosis of streptococcal disease, can solve the problems of lack of diagnostic detection methods, inability to accurately diagnose diseases, and difficult operation techniques

Inactive Publication Date: 2017-02-22
GUANGXI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Streptococcus agalactiae ( Streptococcus agalactiae ) is a highly virulent Gram-positive bacterium that can infect a variety of freshwater and seawater fish, and brings hundreds of millions of losses to the world's aquaculture industry every year. In recent years, tilapia farming in southern my country Due to the lack of effective diagnostic and detection methods in production, tilapia streptococcus agalactiae disease has not been effectively monitored and controlled, causing heavy losses to tilapia farming and seriously hindering the tilapia farming industry in my country development of
At present, the diagnostic and detection methods for Streptococcus agalactiae mainly include: (1) traditional methods such as conventional morphological culture identification, physiological and biochemical identification, etc. These traditional diagnostic detection methods are usually low in accuracy and time-consuming and labor-intensive There are some molecular biological methods such as PCR and LAMP. Although these molecular biological methods have high sensitivity, they are difficult to operate and use at the grassroots level.
(3) Immunological detection methods. There are many immunological detection methods, including antibody detection and antigen detection. Antibody detection methods such as indirect enzyme-linked immunosorbent assay, this method can only detect circulating antibodies, but cannot distinguish current Accurate diagnosis of blight cannot be made

Method used

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  • Double-antibody sandwich enzyme-linked immunosorbent assay kit for detection of Streptococcus agalactiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] In this example, the following process steps were used to prepare a double-antibody sandwich ELISA kit for detecting Streptococcus agalactiae.

[0100] (1) Preparation of multi-well plates coated with 3A9 monoclonal antibody

[0101] Dilute the 3A9 monoclonal antibody to a concentration of 10.0 μg / mL with carbonate buffer, i.e. the coating solution, then add the 3A9 monoclonal antibody dilution into each well of the multi-well plate, place at 4°C for coating At least 12 hours, after the coating time expires, add the skimmed milk powder solution with a mass concentration of 5% into each well of the porous plate, and conduct a blocking reaction at 37°C. The blocking reaction time is at least 1 hour. After the blocking reaction is completed, wash with The buffer solution washes the multi-well plate, and the multi-well plate coated with the 3A9 monoclonal antibody is obtained after the unreacted substances on the multi-well plate are removed, and the storage temperature is ...

Embodiment 2

[0127] The difference from Example 1 is that the critical value of yin and yang (OD 450 average value) as the standard for judging the presence or absence of Streptococcus agalactiae in the test sample.

[0128] In this example, the following process steps were used to prepare a double-antibody sandwich ELISA kit for detecting Streptococcus agalactiae.

[0129] (1) Preparation of multi-well plates coated with 3A9 monoclonal antibody

[0130] Dilute the 3A9 monoclonal antibody to a concentration of 10.0 μg / mL with carbonate buffer, i.e. coating solution (same as above), and then add the 3A9 monoclonal antibody dilution into each well of the multi-well plate, place in 4 ℃ for at least 12 hours, after the expiration of the coating time, add skimmed milk powder solution with a mass concentration of 5% into each well of the multi-well plate, and conduct a blocking reaction at 37°C, the blocking reaction time is at least 1 hour, after the blocking reaction is completed , wash the ...

Embodiment 3

[0158] The difference with embodiment 1 and embodiment 2 is to use negative and positive critical value (OD 450 average value) to judge the specific detection effect on Streptococcus agalactiae.

[0159] According to the detection method of Streptococcus agalactiae double-antibody sandwich enzyme-linked immunosorbent assay kit described in Example 1, Streptococcus agalactiae, Vibrio anguillarum ( Vibrio anguillarum ), Aeromonas hydrophila ( A.hydrophila ), Pseudomonas fluorescens ( P. Fluorescens ), Monas caviae ( Aeromonas caviae ), Pseudomonas aeruginosa ( P. Aeruginosa ) and Pseudomonas stutzeri ( Pseudomonas stutzer ) and SP2 / 0 cells (myeloma cells) were used as a negative control for specificity verification testing, the results are shown in Table 3.

[0160] Table 3 Test results

[0161] Bacteria concentration (CFU / ml) 1.5×10 8

[0162] As can be seen from the results in Table 3, the OD of Streptococcus agalactiae 450 The value is 1.021±0.072, wh...

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Abstract

The invention discloses a double-antibody sandwich enzyme-linked immunosorbent assay (double-antibody sandwich ELISA) kit for detection of Streptococcus agalactiae. The kit is composed of a perforated plate coated with 3A9 monoclonal antibody, a horse radish peroxidase labeled monoclonal antibody 4C12 and a chromogenic substrate 3'.3'.5,5'-tetramethyl benzidine (TMB). The preparation method of the kit includes the steps of: (1) preparing the perforated plate coated with 3A9 monoclonal antibody; (2) preparing the horse radish peroxidase labeled monoclonal antibody 4C12; and (3) preparing the chromogenic substrate 3'.3'.5,5'-tetramethyl benzidine.

Description

technical field [0001] The invention belongs to the technical field of immunodiagnosis and detection of streptococcal disease, and relates to a double-antibody sandwich enzyme-linked immunosorbent assay for streptococcus, that is, Streptococcus agalactiae, and a preparation method of a kit thereof. [0002] technical background [0003] Streptococcus agalactiae ( Streptococcus agalactiae ) is a highly virulent Gram-positive bacterium that can infect a variety of freshwater and seawater fish, and brings hundreds of millions of losses to the world's aquaculture industry every year. In recent years, tilapia farming in southern my country Due to the lack of effective diagnostic and detection methods in production, tilapia streptococcus agalactiae disease has not been effectively monitored and controlled, causing heavy losses to tilapia farming and seriously hindering the tilapia farming industry in my country development of. At present, the diagnostic and detection methods for St...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/535
CPCG01N33/535G01N33/56944G01N33/577
Inventor 陈汉忠梁万文李旻王凯陈明吴文德王瑞
Owner GUANGXI UNIV
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