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
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Examples
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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PUM
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Description
Claims
Application Information
- IPC
- G01N33/531; G01N33/53; G01N33/573
- Inventors
- 马艳平; 柯浩