Method for screening single chain antibodies of Microcystin-LR and verification thereof

A microcystin and single-chain antibody technology, which is applied in botany equipment and methods, biochemical equipment and methods, and the use of vectors to introduce foreign genetic material, etc., can solve the problem of low detection signal of identification methods, interference of culture medium matrix, and screening Single method and other problems, to achieve the effect of strong anti-matrix interference, short screening cycle, fast and easy separation

Inactive Publication Date: 2010-10-13
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a microcystin LR single-chain antibody screening method that is single and inefficient, the detection signal of the identification method is low, and is greatly interfered by the culture medium matrix. The screening and identification method of LR's single-chain antibody, the single-chain antibody also has the potential to be applied in the detection, purification and enrichment of microcystin in water and biological samples

Method used

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  • Method for screening single chain antibodies of Microcystin-LR and verification thereof
  • Method for screening single chain antibodies of Microcystin-LR and verification thereof
  • Method for screening single chain antibodies of Microcystin-LR and verification thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Example 1. The first round of screening of microcystin LR single chain antibody

[0018] During the two rounds of affinity screening in the present invention, the single-chain antibody gene of the phage contained in the human synthetic antibody library is inserted into the coding sequence of the PIX protein gene of the phage, and the single-chain antibody is fused with the PIX protein when expressed On the phage capsid protein, it is displayed and used for affinity screening of antibody libraries.

[0019] (1) Wash magnetic beads: Take 100 μl immunomagnetic beads (provided by Invitrogen, the same below) and 900 μl TBST (TBS buffer containing 1% Tween20), add them to a 1.5ml centrifuge tube and mix well, place the centrifuge tube on a magnetic Let stand on the rack for 1-2 minutes until the mixture becomes clear and the magnetic beads are all adsorbed on the inner wall of the centrifuge tube close to the magnetic rack, then remove the supernatant with a pipette. Then ad...

Embodiment 2

[0022] Example 2. The second round of screening of microcystin LR single chain antibody

[0023] The phage stock solution prepared after the first round of screening was used for the second round of screening, and the amount of phage input in this round of screening was 2 × 10 11 phage (same as Example 1). First, adopt the same method as the first round of screening to complete the steps of magnetic bead washing, coating magnetic beads and negative screening, and then mix the first round of phage stock solution after negative screening with 5 pmol of biotinylated microcystin LR, after incubating with rotation at room temperature for 10 min, add 50 μl of washed avidin-labeled magnetic beads, and continue to incubate with rotation at room temperature for 5 min. The magnetic beads were separated by the magnetic stand, and the magnetic beads were washed twice with TBST containing 0.5% Tween20, and 50 μl of 60 μg / ml trypsin was added to each tube, and the shaker was shaken at a lo...

Embodiment 3

[0027] Example 3. Non-competitive time-resolved fluorescent immunoassay method, phage immunoassay was performed on the initial antibody library and the phage stock solution after two rounds of screening.

[0028] The specific steps of the non-competitive time-resolved fluorescent immunoassay are as follows:

[0029] (1) Wash the avidin-coated ELISA plate once with washing buffer.

[0030] (2) Add 100 μl of biotinylated microcystin to each well of the microplate. Incubate on a shaker at 700rpm for 0.5h at room temperature. Wash the plate 4 times with wash buffer.

[0031] (3) Dilute the original antibody library and the phage stock solution after two rounds of screening, and the diluted phage titer is 10 10 tfu / 100 μl. 100 μl / well was added to the microtiter plate, and each sample was set in 3 replicates, and 3 wells without phage samples were left as blank control wells. Incubate on a shaker at 700 rpm for 1 h at room temperature. Wash the plate 4 times with wash buffer....

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Abstract

The invention relates to a method for screening single chain antibodies of Microcystin-LR and verification thereof. The method comprises the following steps: carrying out two rounds of affinity screening on the biotinylated Microcystin-LR in a human source synthetic antibody library by using an avidin labeled magnetic bead and a negative screening method; extracting total plasmid DNA from phage colonies produced in the second round, carrying out enzyme digestion with enzyme Sfi I, recycling gel to obtain single chain antibody genes, connecting the single chain antibody genes with soluble expression carrier pAK100CL which is processed by enzyme digestion in the same way, and electrically transforming the connected carrier into colibacillus XL1-Blue to obtain soluble expression single chain antibodies; and verifying the soluble expression single chain antibodies by using a competitive time-resolved fluorescence immune analytical method. The invention has the advantage of quick, simple and convenient screening, and can well expose the Microcystin-LR three-dimensional structure into the incubation system. The verification on the screening result has the advantages of high detection signal and strong anti-interference capacity against stroma.

Description

Technical field: [0001] The invention relates to a screening method and identification of a single-chain antibody of microcystin LR. Background technique: [0002] Microcystins are common algal toxins in eutrophic freshwater, and they are a class of cyclic heptapeptides with protein phosphatase inhibitory and hepatocarcinogenic effects. More than 60 types of microcystins have been reported so far, among which microcystin LR is known to be the most toxic and acutely harmful freshwater cyanotoxin. In 1998, the World Health Organization (WHO) added a guideline for microcystin LR to 1ppb in the guidance of drinking water standards. my country's current drinking water quality standards also include the detection items of microcystin LR. [0003] At present, there have been a few reports on the use of phage display technology to screen and prepare single-chain antibodies or polypeptide ligands of microcystin LR. McElhiney et al[Jacqui McElhiney, MathewDrever, Linda A.Lawton, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C12N15/70C07K16/14G01N33/53
Inventor 刘媛刘贤金梁颖张存政王耘温爽
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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