Monoclonal antibody identifying whether goat milk is mixed with cow milk or not through specificity and preparation method thereof

Monoclonal antibody identifying whether goat milk is mixed with cow milk or not through specificity and preparation method thereof

  • CN 104,725,508 A
  • Filed: 04/14/2015
  • Published: 06/24/2015
  • Est. Priority Date: 04/14/2015
  • Status: Active Application
First Claim
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1. specificity differentiates the preparation method of the monoclonal antibody of sheep Ruzhong admixture cow'"'"'s milk, it is characterized in that:

  • Comprise the following steps;

    Step one;

    prepare immunizing antigen;

    Get fresh milk and be placed in 4 DEG C of whizzers, 4000 ~ 6000rpm, 10 ~ 20min is centrifugal;

    Cow'"'"'s milk layering, upper strata is mainly fat, discards completely, retains and fully mixes remainder, being skimming milk, as immunizing antigen;

    Step 2;

    immunity;

    Get 6-8 female BAl BIc/c mouse in age in week, utilize immunizing antigen immunity 4 times, immunity terminates to carry out cytogamy after three days;

    Step 3;

    prepare hybridoma;

    Cytogamy the day before yesterday, preparation feeder layer cells;

    Cytogamy is carried out under aseptic condition, by be separated splenocyte with SP2/0 oncocyte in (5-10);

    the ratio of 1 mixes, under the effect of PEG1500, carry out cytogamy, by fusion after hybridoma be laid in the 96 porocyte culture plates containing feeder layer cells, be placed in 37 DEG C, 5% CO 2cultivate in incubator;

    Step 4;

    screening hybridoma;

    Conveniently indirect ELISA detects Hybridoma Cell Culture supernatant;

    use fresh cow milk respectively, fresh sheep breast, bovine whey is as envelope antigen, 200ul/ hole, wrap rear, 4 DEG C are spent the night, PBST washs 3 times, add cells and supernatant, using negative mice serum as negative control, 100ul/ hole, each extent of dilution does 3 repetitions, hatch 1 ~ 2h for 37 DEG C, PBST washs 3 times, add the antibody of horseradish peroxidase-labeled, 1;

    5000 dilutes, 100ul/ hole, hatch 1h for 37 DEG C, PBST washs 3 times, add TMB nitrite ion, hatch 20min for 37 DEG C, add stop buffer, be placed in microplate reader and detect each hole OD 450value, with S/P>

    2.1 for positive judging criterion, marks and screens the cell culture well containing positive hybridoma cell, Carry out mono-clonal cultivation by screening the positive hybridoma cell obtained, mono-clonalization is cultivated and is adopted limiting dilution assay;

    first, carry out cell counting, according to cell counts, dilute cell suspension;

    Getting 230 viable cell is suspended in the RPMI1640 substratum of 4.6ml, and now, average every 0.1ml solution contains 5 cells, is inoculated in 96 orifice plates, every hole 0.1ml, totally 36 holes;

    Residue 1ml, then add 4mlRPMI1640 substratum, 5ml altogether, now, average every 0.1ml solution, containing 1 cell, is inoculated in 36 holes of next, every hole 0.1ml;

    Finally remain 1.4ml, then add 1.4ml, 2.4ml, is inoculated in residue 24 hole, every hole 0.1ml altogether, now every 0.5, hole cell;

    After bed board terminates, under being placed in microscope, mark the cell culture well only containing a cell;

    The hybridoma stably excreting monoclonal antibody obtained, can non-whey antigen in specific recognition cow'"'"'s milk, and there is not cross reaction with sheep breast.

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