国产沙门氏菌免疫磁珠的制备及其应用

目的优化免疫磁珠制备条件,获得捕获性能高、成本低的国产沙门氏菌免疫磁珠,为国产沙门氏菌免疫磁珠的产业化制备奠定基础。方法采用亚微米级羧基磁珠及沙门氏菌多克隆抗体,利用碳二亚胺缩合法制备2 mg级免疫磁珠。以目标菌捕获率、磁珠表面抗体偶联率为评价指标,对4种粒径(100、300、500、1000 nm)的磁珠原材料进行筛选;以目标菌捕获率、磁珠表面抗体偶联率、免疫磁珠表面FITC荧光强度为评价指标,对磁珠与抗体不同的投料比(100:1、100:1.5、100:2、100:2.5、100:3)进行优化;同时,对国产沙门氏菌免疫磁珠与进口免疫磁珠(Dynalbeads~?沙门氏菌免疫磁珠、Bac Trace~?沙门氏菌免疫磁珠)的性能进行比较分析,考察了捕获率、免疫磁珠与目标菌的结合情况、特异性捕获能力等,并在实际样品中进行了应用检测。结果以粒径为300 nm的磁珠为原材料制备的免疫磁珠对浓度为10~2 CFU/mL的目标菌的捕获率为99%、抗体偶联率为34.64%,明显高于其他粒径的免疫磁珠;当投料比为100:2(磁珠:抗体)时,制备的免疫磁珠对浓度为10~2 CFU/m L的目标菌的捕获率为100%,偶联率为52.34%,优于其他投料比;针对不同浓度的鼠伤寒沙门氏菌(10~0~10~6 CFU/mL),国产免疫磁珠的捕获率高于进口免疫磁珠(国产:83%~100%;Dynalbeads~?:57%~74%;Bac Trace~?:9%~40%);在25 mL牛奶中添加(11.9±3.7)CFU鼠伤寒沙门氏标准菌株,国产沙门氏菌免疫磁珠的检出率高于进口免疫磁珠(国产:100%;Dynalbeads~?:70%;Bac Trace~?:40%)。结论优化了制备条件后所制备的国产沙门氏菌免疫磁珠的捕获能力强、价格低、能够实现大体积实际样品中痕量目标菌的100%检出,具有广阔的应用前景,本研究为沙门氏菌免疫磁珠的国产化应用提供了科学数据。

Preparation and application of domestic anti-Salmonella immunomagnetic beads

Objective To optimize the preparation conditions of anti-Salmonella immunomagnetic beads (IMBs), obtain anti-Salmonella IMBs with high capture ability, low preparation cost and provide the foundation for the industrialization of domestic anti-Salmonella IMBs. Methods IMBs (2 mg) were prepared with sub-micron carboxyl beads and Salmonella polyclonal antibodies by carbodiimide condensation method. Four IMBs diameter (100, 300, 500, 1000 nm) were screened by capture efficiency (CE) and rate of antibody conjugate; the magnetic bead-antibody mass ratio (100:1, 100:1.5, 100:2, 100:2.5, 100:3) were optimized base on the CE, rate of antibody conjugate and FITC fluorescence intensity on the surface of the IMBs. Besides, the capacity of domestic anti-Salmonella IMBs and imported IMBs (Dynalbeads? anti-Salmonella beads, Bac Trace? anti-Salmonella CSA-1 magnetic beads) were analyzed contrastively by CE, combination of IMBs with target bacteria and specificity. IMBs were also evaluated in detection of practical sample. Results The CE of IMBs with 300 nm magnetic beads was 99% for 102 CFU/mL Salmonella and the rate of antibody conjugation was 34.64%, higher than that with other diameters. The CE of IMBs which prepared with 100:2 of mass ratio was 100% and the rate of antibody conjugation was 52.34%. For different concentrations of Salmonella typhimurium (100~106 CFU/mL), the CE of domestic anti-Salmonella IMBs was higher than that of imported IMBs (domestic anti-Salmonella IMBs: 82~100%; Dynalbeads? anti- Salmonella beads: 57%~74%; Bac Trace? Anti-Salmonella CSA-1 magnetic beads: 9%~40%). For 25 mL milk with (11.9±3.7) CFU Salmonella Typhimurium, the detection rate of domestic anti-Salmonella IMBs was higher than that of imported IMBs (domestic anti-Salmonella IMBs: 100%; Dynalbeads? anti- Salmonella beads: 70%; Bac Trace? Anti-Salmonella CSA-1 Magnetic Beads: 40%). Conclusion The optimized domestic IMBs have strong capture ability, low price, 100% detection rate for low level Salmonella Typhimurium in high volume practical sample. In this study, the optimized preparation conditions of anti-Salmonella IMBs have high application value and provide a precondition for preparation and application of domestic Salmonella IMBs.