Immunoaffinity chromatographic column used for purifying melamine and its method for purifying melamine
A melamine, immunophilic technology, applied in measuring devices, instruments, scientific instruments, etc., can solve problems such as non-commercialized IAC columns, and achieve the effect of reducing analysis costs and environmental pollution, simple operation, and maintaining activity.
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Embodiment 1
[0026] Example 1 Preparation of melamine monoclonal antibody
[0027] Animal immunization: melamine was used as hapten, coupled with carrier protein BSA as immunogen by glutaraldehyde method, BALB / C mice were selected as immunized animals, and the immunization dose was 100 μg / mouse, and an equal volume of complete Fumidine was mixed for the first immunization After 21 days, an equal volume of incomplete Freund's adjuvant was added for booster immunization at the same dose, and blood was collected on the 10th day after the second immunization for testing, and then a booster immunization was performed every 21 days until the serum titer and specificity If the sex meets the requirements, then the immunization dose is halved for sprint immunization, and splenocytes are taken for fusion 3 days later.
[0028] Cell fusion: feeder cells were prepared the day before fusion, and on the day of fusion, splenocytes were fused with SP2 / 0 myeloma cells at a ratio of 5:1.
[0029] Hybri...
Embodiment 2
[0032] Example 2 Preparation of Immunoaffinity Chromatography Column (IAC)
[0033]The specific steps are as follows: mix 0.2mL, 0.04 mol / L HCL, 0.75mL deionized water and 3.4mL tetramethoxysilane TMOS, and store at 4°C for 2-3min. The temperature of the above TMOS solution was controlled at about 0°C and ultrasonically mixed for 30 min. Take a beaker, weigh it, draw a certain amount of antibody solution, dilute it to 1mg / mL with 0.01M, pH7.4 PBS buffer, and store it at 4°C. After the TMOS solution is sonicated completely, draw 1 mL of it and add it to the antibody solution stored at 4°C to form a gel. After the gel is finished, weigh the beaker again and place it in a constant temperature oven at 37°C for gel aging. When the gel loses 50% of its initial weight, the aging process ends. Grind the aged gel (about 1g) and pack it into a column. After the gel deposition in the column is stable, pre-wash with 5mL 0.01M, pH7.4 PBS to wash away unembedded antibodies and other int...
Embodiment 3
[0034] The determination of embodiment 3 IAC column capacity:
[0035] Take out the IAC column prepared in Example 2, remove the plugs at the upper and lower ends of the column, connect the column to a vacuum pump, adjust the flow rate so that the liquid flows out at a rate of 2d / s (not exceeding 1mL / min), and after the liquid is drained, Load 10mL, 10μg / mL treated sample solution (sample dissolved in loading buffer) at the same flow rate as above, collect the loading filtrate, and measure OD 240 . After the liquid is drained, rinse with 10mL of 0.01M, pH7.4 PBST and 10mL of pure water, respectively, at the same flow rate as above. Collect the wash filtrate and measure the OD 240 . as the OD 240 If the value is greater than 0.02, repeat the washing until it is less than 0.02, and calculate the volume of the washing filtrate and the content of melamine in the filtrate. After the liquid is drained, load 1mL of 0.01M glycine hydrochloride solution containing 50% methanol, pH...
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