A sensitive sandwich enzyme immunoassay for human pulmonary surfactant protein D (SP-D) was developed and used to examine the blood SP-D levels of drowning victims. Human SP-D was purified from amniotic fluid by chromatographic methods, and an antibody against human SP-D was prepared. A polystyrene ball coated with anti-SP-D IgG was incubated with purified human SP-D, and then with anti-SP-D Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as the hydrogen donor. The detection limit of human SP-D was 5.2 pg per assay tube. Examination of cross-reactions of this sandwich enzyme immunoassay with proteins from other human organs showed it to be highly specific for lung, and Northern blot analysis detected specific SP-D mRNA expression only in lung. The SP-D concentration of normal human serum was 6.4+/-2.7 (mean+/-S.D.) ng ml(-1) (n=20). The recovery rates of 0.52 ng and 5.2 ng SP-D added to 5 microl normal human serum were 93.6+/-2.7% and 93.6+/-6.1%, respectively. Blood SP-D levels of victims from the saltwater drowning group (n=14) revealed higher concentrations (105.8+/-53.7 ng ml(-1)), while freshwater drowning victims (n=12) were estimated to be 74.1+/-43.9 ng ml(-1). The SP-D levels of 15 subjects who died of hemorrhage (n=5), heart failure (n=8), traumatic shock (n=1), and electrocution (n=1) were lower (22.0+/-8.5 ng ml(-1)), and those of asphyxia victims (n=10) were slightly higher (36.2+/-17.1 ng ml(-1)) than those of other causes of death, except for drowning. These results suggest that in drowning victims, SP-D flowed into the systemic circulation by physiological and physical mechanisms, and the differences of blood SP-D levels between saltwater drowning and freshwater drowning victims are presumed to be influenced by the type of agony and/or the length of survival time in water.

译文

:开发了一种针对人肺表面活性物质D(SP-D)的敏感三明治酶免疫测定法,并用于检查溺水受害者的血液SP-D水平。通过色谱法从羊水中纯化出人SP-D,并制备了针对人SP-D的抗体。将涂有抗SP-D IgG的聚苯乙烯球与纯化的人SP-D孵育,然后与抗SP-D Fab'-过氧化物酶偶联物孵育。使用3-(4-羟苯基)丙酸作为氢供体,通过荧光分析法测定了与聚苯乙烯球结合的过氧化物酶活性。每个分析管中人SP-D的检出限为5.2 pg。对该夹心酶免疫测定法与其他人体器官蛋白质的交叉反应的检查表明,它对肺具有高度特异性,Northern印迹分析仅在肺中检测到特定的SP-D mRNA表达。正常人血清的SP-D浓度为6.4 /-2.7(平均值/-S.D。)ng ml(-1)(n = 20)。添加到5微升正常人血清中的0.52 ng和5.2 ng SP-D的回收率分别为93.6 /-2.7%和93.6 /-6.1%。盐水淹没组(n = 14)受害者的血液SP-D水平显示较高的浓度(105.8 /-53.7 ng ml(-1)),而淡水淹没的受害者(n = 12)估计为74.1 /-43.9 ng ml(-1)。 15名死于出血(n = 5),心力衰竭(n = 8),外伤性休克(n = 1)和电击伤(n = 1)的受试者的SP-D水平较低(22.0 /-8.5 ng ml (-1)),窒息受害者(n = 10)的死亡人数(36.2 /-17.1 ng ml(-1))略高于其他死亡原因,但溺水除外。这些结果表明,在溺水受害者中,SP-D是通过生理和物理机制流入全身循环的,据推测,海水淹没受害者与淡水溺水受害者之间血液SP-D水平的差异受痛苦和//类型的影响。或在水中生存时间的长短。

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