Preparation method and applications of biotin marked rabbit anti-tilapia IgM (immunoglobulin m) polyclonal antibody

A polyclonal antibody and biotin-labeled technology, which is applied in biological testing, material inspection products, measuring devices, etc., to achieve the effects of high titer, good specificity, important theoretical significance and production value

Active Publication Date: 2013-12-18
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no rabbit anti-tilapia IgM antibody on the market, which provides unfavorable factors for the monitoring of tilapia immune response level and the establishment of immune detection methods

Method used

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  • Preparation method and applications of biotin marked rabbit anti-tilapia IgM (immunoglobulin m) polyclonal antibody
  • Preparation method and applications of biotin marked rabbit anti-tilapia IgM (immunoglobulin m) polyclonal antibody

Examples

Experimental program
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Embodiment 1

[0030] The preparation method of the rabbit anti-tilapia IgM polyclonal antibody provided in this embodiment, the steps are as follows:

[0031] (1) 5 healthy tilapias, weighing 400-450g, blood was collected from the tail artery, placed at 37°C for 2 hours, left at 4°C overnight to fully analyze the serum, centrifuged at 4000rpm / min at 4°C for 30min, and the supernatant was taken;

[0032] (2) Dilute the tilapia serum with an equal volume of 0.01mol / L PBS (pH7.4), add twice the saturated ammonium sulfate of pH7.4, and stir while adding the solution; continue stirring for 1 hour after adding, and carry out Centrifuge, 7000rpm, 30min, take the precipitate;

[0033] (3) After dissolving the precipitate with PBS, the ProteinG+ProteinA affinity chromatography column was used to purify IgM, and the BCA kit was used to measure the concentration, such as figure 1 Shown is the IgM SDS-PAGE pattern after purification;

[0034] (4) Mix 200 μg of purified tilapia IgM with an equal volum...

Embodiment 2

[0042] The application steps of the biotin-labeled rabbit anti-tilapia IgM polyclonal antibody provided in this example are as follows:

[0043] (1) The tested tilapia was 1.93×10 3cfu / tail low-dose Streptococcus agalactiae intraperitoneal injection, collect 48h, 72h, one week, 12 days of tail artery blood, 37 ℃ for 2 hours, 4 ℃ refrigerator overnight to fully analyze the serum, centrifuge at 4000rpm / min for 10min, take the supernatant , get tilapia serum;

[0044] (2) Take Streptococcus agalactiae bacteria liquid, centrifuge at 12000rpm / min for 5min, ultrasonically crush, measure the concentration of lysed bacteria liquid with BCA kit, coat 100μl / well with 80μg / ml concentration, and coat 100μL / well with ELISA plate, overnight at 4°C ;

[0045] (3) Shake off the coating solution, block the ELISA plate with 1% bovine serum albumin, and act for 2 hours at 37°C;

[0046] (4) Wash the plate three times with PBST, gradiently dilute the serum obtained in step (1) with PBS solutio...

Embodiment 3

[0052] The rabbit anti-tilapia IgM polyclonal antibody provided in this example is applied to Western-blot, and the steps are as follows:

[0053] (1) 5 μg of tilapia IgM was subjected to conventional SDS-PAGE electrophoresis with 12% separating gel;

[0054] (2) After electrophoresis, transfer to nitrocellulose membrane (NC);

[0055] (3) After the membrane transfer, cut off the Marker band and stain it in the amino black staining solution for 5 minutes, and then decolorize it with the decoloring solution until the protein band is clear;

[0056] (4) The NC membrane was added to 5% skimmed milk powder, sealed at 37°C for 1 hour, washed 3 times with TBST, 10 minutes each time;

[0057] (5) Add purified polyclonal antibody (diluted to 3.125 μg / mL), incubate at 37°C for 60 minutes, wash with TBST 3 times, 10 minutes each time;

[0058] (6) Add fluorescently labeled Donkey anti-Rabbit IgG1:15000, incubate at 37°C for 60 minutes, wash with TBST 3 times, 10 minutes each time;

...

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Abstract

The invention discloses a preparation method of a biotin marked rabbit anti-tilapia IgM (immunoglobulin m) polyclonal antibody. The preparation method comprises the following steps: collecting tilapia serum, and purifying to obtain purified tilapia IgM; immunizing a rabbit by using the tilapia IgM to obtain a rabbit anti-tilapia IgM polyclonal antibody, and detecting the titer of anti-tilapia serum of the rabbit anti-tilapia IgM polyclonal antibody by adopting ELISA (enzyme linked immunosorbent assay); strengthening the immunity of the purified tilapia IgM, then collecting and separating to obtain serum containing the rabbit anti-tilapia IgM polyclonal antibody, purifying, adding biotin and mixing uniformly to obtain the biotin marked rabbit anti-tilapia IgM polyclonal antibody. The biotin marked rabbit anti-tilapia IgM polyclonal antibody prepared by the method disclosed by the invention is good in specificity and high in purity and titer, and can have a specific binding reaction with the tilapia IgM. The invention also discloses applications of the biotin marked rabbit anti-tilapia IgM polyclonal antibody prepared by the method in ELISA and Western-Blot.

Description

technical field [0001] The invention belongs to the technical field of polyclonal antibodies, in particular to a preparation method and application of a biotin-labeled rabbit anti-tilapia IgM polyclonal antibody. Background technique [0002] Immunoglobulin is an important molecule in the humoral immune response of animals, and the main immunoglobulin of teleosts as lower vertebrates is IgM. The molecule is a tetramer containing 2 heavy chains and 2 light chains per monomer. Highly purified fish serum IgM has important uses in fish immunology, such as preparing monoclonal and polyclonal antibodies, detecting fish pathogens and fish immune response levels; exploring the production and distribution of IgM, and can also be used to study immune System evolution. [0003] Tilapia has been listed as a high-quality fish species for industrial development by the Ministry of Agriculture. In recent years, with the destruction of the breeding environment and the increase of breeding ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/53G01N33/573
Inventor 马艳平柯浩刘振兴冯国清郝乐
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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