Method for improving stability of tuberculin BCG-PPD skin test diagnostic reagent
A diagnostic reagent, a technology for tuberculin, which is applied in the field of improving the stability of tuberculin BCG-PPD skin test diagnostic reagents, can solve the problems of short shelf life of the product and cannot meet the actual market demand, and achieves good stability and a broad market. Prospect and application value, the effect of high detection sensitivity
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Embodiment 1
[0050] Example 1 bacterial culture
[0051] (1) 1 seed of recovery work was inoculated into 5 Roche's egg culture medium, and cultured for 2 weeks. After the bacteria grew into dry, wrinkled and slightly light yellow bacterial film, the growth of the bacterial lawn could be observed at this time: the end of the culture After that, each 1st-generation strain was first suspended with 5ml of sodium chloride 0.9% solution, collected all the bacterial liquid, and sampled for acid-fast staining. Acid-fast staining was positive (bacteria were stained red). Biochemical reaction: Nitrate reduction reaction is positive, resulting in red; urease reaction is positive, showing red; heat-resistant catalase reaction should be negative, no bubbles are generated in 10-20 minutes; polysorbate 80 hydrolysis reaction does not change color. Each medium tube was resuspended with 2ml of physiological saline to prepare a bacterial solution, and a total of 10ml (1st generation) was collected. The bac...
Embodiment 3
[0065] Example 3 Precipitation
[0066] A 40% trichloroacetic acid solution was prepared, added to the harvest solution at a ratio of 1:10, with a final concentration of 4%, and allowed to stand at room temperature for 2 hours. After shaking well, centrifuge at 4°C and 8000rpm for 20min. The precipitate was collected and weighed 450 g.
[0067] Precipitate 450g is dissolved with 3150ml of 0.9% sodium chloride solution, adjusts the pH value with 1M sodium hydroxide, adds ammonium sulfate according to the amount of precipitation dissolution, makes its concentration reach 60% saturation. Add 390g×3.15L=1228.5g of ammonium sulfate, let stand at 2-8°C overnight (16 hours), centrifuge at 8000rpm for 20min at 4°C, and collect 365g of precipitate. Add 2555ml of 0.9% sodium chloride solution to dissolve the precipitate. Add 40% trichloroacetic acid to a final concentration of 4%, precipitate protein, centrifuge at 8000 rpm, collect the precipitate, and weigh 310 g. The precipitatio...
Embodiment 5
[0070] Embodiment 5 sterile filtration
[0071] The dialysate was filtered and clarified through a 1.2μm-0.45μm microporous membrane, and after filtration, a 10ml sample was taken to determine the microbial limit, and then sterilized and filtered through a 0.45μm-0.22μm filter membrane to obtain the stock solution, and a total of 1800ml was harvested. The sterilization and filtration time should be controlled within 4 hours. The stock solution was prepared according to the protein concentration of 9mg / ml / , and then lyophilized for storage.
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