Chip detection method of visual protein in residual gentamicin in animal derived food
A gentamicin, animal-derived technology, applied in the field of visual protein chip detection, can solve the problems of being unsuitable for on-site monitoring and screening of a large number of samples, high requirements for technicians, and high equipment requirements, to achieve visualization of test results, reduce Operation steps, high-sensitivity effects
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Embodiment 1
[0048] Example 1: Preparation of gentamicin-coated antigen.
[0049] 1) Dissolve 5 mg of GM in 10 mL of PBS buffer (0.1 M, pH 7.4) to obtain solution I, and stir in an ice-water bath for 10 min.
[0050] 2) Dissolve 20 mg of carbodiimide in 10 mL of PBS buffer (0.1 M, pH 7.4) to obtain solution II, and stir in an ice-water bath for 10 min.
[0051] 3) Weigh 10 mg of carrier protein and dissolve in 20 mL of PBS buffer (0.1 M, pH 7.4) to obtain solution III, and stir in an ice-water bath for 10 min.
[0052] 4) Add solution I to solution III and stir on a magnetic stirrer, then slowly add solution II to the above mixture drop by drop in an ice-water bath, stir for 1 hour in the dark, and react for 3 hours at 4°C to synthesize gentamicin Protein-carrier protein coupling product.
[0053] 5) Put the gentamicin-carrier protein coupling product into a dialysis bag, dialyze with PBS buffer (0.1M, pH7.4) at 4°C for 3 days, and change the dialysate every 8 hours.
[0054] 6) The cou...
Embodiment 2
[0055] Example 2: Preparation of gentamicin-coated antigen.
[0056] 1) Dissolve 200mg GM in 50mL PBS buffer (0.1M, pH7.4) to obtain solution I, and stir in an ice-water bath for 10min.
[0057] 2) Dissolve 100 mg of carbodiimide in 50 mL of PBS buffer (0.1 M, pH 7.4) to obtain solution II, and stir in an ice-water bath for 10 min.
[0058] 3) Weigh 50 mg of carrier protein and dissolve in 100 mL of PBS buffer (0.1 M, pH 7.4) to obtain solution III, and stir in an ice-water bath for 10 min.
[0059] 4) Add solution I to solution III and stir on a magnetic stirrer, then slowly add solution II to the above mixture drop by drop in an ice-water bath, stir for 1 hour in the dark, and react for 3 hours at 4°C to synthesize gentamicin Protein-carrier protein coupling product.
[0060] 5) Put the gentamicin-carrier protein coupling product into a dialysis bag, dialyze with PBS buffer (0.1M, pH7.4) at 4°C for 3 days, and change the dialysate every 8 hours.
[0061] 6) The coupled pr...
Embodiment 3
[0062] Example 3: Method 1 for the quantitative detection of gentamicin content by visual protein chip method.
[0063] (1) Using bovine serum albumin (BSA) as a blank control, the bovine serum albumin and gentamicin-carrier protein conjugates were spotted by a biochip spotter according to different specifications of the matrix, and the wells of the spot plate were Add 30 μL of bovine serum albumin and 30 μL of gentamicin-carrier protein conjugate, and place the spotted slides in a 4°C refrigerator to fix overnight;
[0064] (2) After fixation, add 20 μL of 10 mg / mL bovine serum albumin buffer solution to each reaction well to block the chip for 1 hour, then wash with PBST 3 times for 5 minutes each time; The area is blocked to prevent non-specific binding of proteins in the sample to be tested and other active groups, resulting in high background or false positives;
[0065] (3) Add 20 μL of gentamicin antibody and different concentrations of free gentamicin standard solutio...
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