金标SPA快速检测IgG含量的方法建立
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摘要
为了建立快速检测血清中IgG含量的金标SPA法,本论文通过金标SPA的制备、金标SPA法的条件优化、评价和应用试验,初步建立了一个快速检测猪血清IgG含量的方法。
金标SPA的制备。本试验用直径为10nm的胶体金对SPA(金黄色葡萄球菌A蛋白)进行标记和超速离心纯化,制得金标SPA即金探针,用两点法检测金标SPA的生物活性和稳定性,结果发现:金标SPA的活性较好,能稳定地保存三个月左右。
金标SPA法的条件优化。本试验分别从金探针的浓度、溶液pH值、温度和作用时间四个因素进行探讨,结果发现在常温(15—20℃)且温度变化不大(±1℃)、pH7.4、金标SPA最佳稀释比为1∶4的条件下作用40min左右为金标SPA法的最佳作用条件;测定猪血清中IgG含量时,血清最佳稀释倍数为1∶1000。
方法的评价和应用。本试验以检测猪血清中IgG为例,通过线性范围试验、批内和批间重复性试验、回收试验、方法对比试验对金标SPA法进行评价,并在成年猪、不同日龄仔猪的血清IgG检测方面进行应用,进一步验证了该法的可行性和实用性。结果表明:该方法灵敏度高,检测范围2.16～116.67μg/mL;回收率为98.18%;批内和批间变异系数分别为4.54%,5.88%;通过与夹心ELASA方法进行对比,发现两种方法的测定值高度相关(r=0.9945),但金标SPA法操作简便、快速、一步完成、不需多次洗涤、易于自动化操作。因此,具有很高临床应用价值。
IThe paper takes detecting the swine blood serum as an example through three parts:SPA labeled with colloidal gold preparation;Optimizing of the method condition;The evaluating experiment of method and applies primarily,establishes one method to detect the IgG content rapidly.
SPA labeled with colloidal gold preparation.This experiment first uses the diameter 10nm colloid gold to label SPA(the golden yellow staphylococcus protein A),with the ultracentrifugation purified SPA -coated gold particles,also called the gold probe,and has carried on the examination to its biological activity and stability by two point method.The results showed that the obtained gold probe activeness is better,and it can be preserved stably for three months or so.
Optimizing of the method condition.From four influencing factors of the gold probe density,the solution PH,the detection temperature and the response time to carry on the discussion,obtains the best detection condition is the best gold probe density is 1:4;The best PH is 7.4;In order to simplify the experiment,We decided to characterize the assaying performance at room temperature(±0.5℃);Reaction time about 40 minutes.Taking the swine blood serum IgG as the example,the best surem dilution multiple is 1:1000.
The evaluating experiment of method and applies primarily.,then through the linear scope experiment,the duplicated experiment,the returns-ratio experiment,the method contrast experiment to evaluate this immunassay.And has carried separately on the examination with the newly built method to the blood serum samples of grow-up swine and the different date age newborn pig has confirmed furtherly that this method is feasibile and useful.The results showed that this method is highly sensitive,the lowest detection limits may reach 2.16μg/mL and the highest detection limits is 116.67μg/mL,the returns-ratio reaches 98.18%;in approves the coefficient of variation CV%=4.54%;among approves CV%=5.88%;Contrasted with the ELASA method,determination value between these two method has the highly correlational dependence,also r=0.9945,but this method operation is simple and fast. one step completed,it does not have to wash in many times,simplifying the automated operation.So it has very high clinical using value.
金标SPA的制备。本试验用直径为10nm的胶体金对SPA(金黄色葡萄球菌A蛋白)进行标记和超速离心纯化,制得金标SPA即金探针,用两点法检测金标SPA的生物活性和稳定性,结果发现:金标SPA的活性较好,能稳定地保存三个月左右。
金标SPA法的条件优化。本试验分别从金探针的浓度、溶液pH值、温度和作用时间四个因素进行探讨,结果发现在常温(15—20℃)且温度变化不大(±1℃)、pH7.4、金标SPA最佳稀释比为1∶4的条件下作用40min左右为金标SPA法的最佳作用条件;测定猪血清中IgG含量时,血清最佳稀释倍数为1∶1000。
方法的评价和应用。本试验以检测猪血清中IgG为例,通过线性范围试验、批内和批间重复性试验、回收试验、方法对比试验对金标SPA法进行评价,并在成年猪、不同日龄仔猪的血清IgG检测方面进行应用,进一步验证了该法的可行性和实用性。结果表明:该方法灵敏度高,检测范围2.16～116.67μg/mL;回收率为98.18%;批内和批间变异系数分别为4.54%,5.88%;通过与夹心ELASA方法进行对比,发现两种方法的测定值高度相关(r=0.9945),但金标SPA法操作简便、快速、一步完成、不需多次洗涤、易于自动化操作。因此,具有很高临床应用价值。
IThe paper takes detecting the swine blood serum as an example through three parts:SPA labeled with colloidal gold preparation;Optimizing of the method condition;The evaluating experiment of method and applies primarily,establishes one method to detect the IgG content rapidly.
SPA labeled with colloidal gold preparation.This experiment first uses the diameter 10nm colloid gold to label SPA(the golden yellow staphylococcus protein A),with the ultracentrifugation purified SPA -coated gold particles,also called the gold probe,and has carried on the examination to its biological activity and stability by two point method.The results showed that the obtained gold probe activeness is better,and it can be preserved stably for three months or so.
Optimizing of the method condition.From four influencing factors of the gold probe density,the solution PH,the detection temperature and the response time to carry on the discussion,obtains the best detection condition is the best gold probe density is 1:4;The best PH is 7.4;In order to simplify the experiment,We decided to characterize the assaying performance at room temperature(±0.5℃);Reaction time about 40 minutes.Taking the swine blood serum IgG as the example,the best surem dilution multiple is 1:1000.
The evaluating experiment of method and applies primarily.,then through the linear scope experiment,the duplicated experiment,the returns-ratio experiment,the method contrast experiment to evaluate this immunassay.And has carried separately on the examination with the newly built method to the blood serum samples of grow-up swine and the different date age newborn pig has confirmed furtherly that this method is feasibile and useful.The results showed that this method is highly sensitive,the lowest detection limits may reach 2.16μg/mL and the highest detection limits is 116.67μg/mL,the returns-ratio reaches 98.18%;in approves the coefficient of variation CV%=4.54%;among approves CV%=5.88%;Contrasted with the ELASA method,determination value between these two method has the highly correlational dependence,also r=0.9945,but this method operation is simple and fast. one step completed,it does not have to wash in many times,simplifying the automated operation.So it has very high clinical using value.
引文
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[13]刘春龙,李忠秋等.免疫胶体金法和免疫单扩散法检测牛初乳中IgG含量的比较研究[J].吉林农业大学学报,2006,4.
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[18]陈凤梅,李娟等.免疫胶体金技术应用及研究进展[J].中国兽药杂志,2004,8,38(8):33-35
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