Phenotypic analysis of fetal blood leucocytes: Potential for prenatal diagnosis of immunodeficiency disorders

Recent technological advances allow the detection and quantitation of subsets of leucocytes using monoclonal antibodies. We have taken advantage of this to study the ontogeny of fetal blood leucocytes, using very small blood samples obtained at fetoscopy. By 14 weeks gestation T cells represent 35 per cent or more of fetal leucocytes and the distribution of the helper/inducer and suppressor/cytotoxic subsets is similar to that of adults. B lymphocytes before 161/2 weeks are low (4–20 per cent), but rise to a mean of 28 per cent in 17–26 week fetuses. Granulocytic cells, many of which are phenotypically immature, represent 18–34 per cent of total leucocytes. The methodology employed is very reliable and offers the opportunity for the prenatal diagnosis of some immunodeficiency disorders, since using the same reagents we have diagnosed children with severe combined immunodeficiency shortly after birth.     

Development of enzyme immunoassay for detection of DDT

Dichlorodiphenyltrichloroethane (DDT) is one of the persistent organic pollutants (POPs) widely found in the environment and in the general population. In this study, a direct competitive enzyme immunoassay (EIA) has been developed for the quantitative analysis of DDT. To generate a specific polyclonal antibody for EIA, p, p′-DDT was conjugated to porcine thyroglobulin for rabbit immunization. At optimized EIA conditions, the standard curves ranged from 0.137 to 100 ng/mL with the quantification limit of 0.41 ng/mL. The coefficients of variation (CV%) were 5.42–10.53% for intra-assay and 6.04–7.26% for inter-assay. Cross-reactivities with DDT metabolites (DDTs, including o, p′-DDT, p, p′-DDD, o, p′-DDD, p, p′-DDE, o, p′-DDE, p, p′-dichlorobenzophenone (DCBP), o, p′-DCBP) were investigated. The polyclonal antibody showed relatively low and/or no cross-reactivity with these compounds, and the assay was seen to be highly selective for p, p′-DDT. Moreover, the DDTs could be ranked by their reactivity: DDT > DDD > DDE > DCBP. In addition, the characterization of the polyclonal antibody indicated that the antiserum possesses a high specificity for p, p′-isomers. The results indicated that the developed EIA using this antibody could be a convenient and supplemental analytical tool for monitoring DDT.     

Amniotic fluid acetylcholinesterase measurements: Comparing immunochemical and polyacrylamide gel techniques

In the present study, a recently reported immunochemical technique for measuring acetylcholinesterase (AChE) in amniotic fluid utilizing the 4F19 antibody was compared with the widely utilized polyacrylamide gel technique to determine whether the immunochemical assay provided an advantage in separating unaffected pregnancies from those associated with open spina bifida (OSB) and open ventral wall defects (OVWD). The study included (1) 73 amniotic fluid samples from unaffected pregnancies [alpha-fetoprotein (AFP) < 2 MoM] with no visible gel AChE band, (2) nine bloodstained samples from unaffected pregnancies (AFP 2·2–4·0 MoM) with visible gel AChE bands, (3) 18 samples associated with OSB (AFP 2·2–7·0 MoM) with visible gel AChE bands, and (4) 20 samples associated with OVWD (AFP 3·2–53·5 MoM) with visible gel AChE bands. The immunochemical assay produced ranges of measurements in the four respective categories as follows: (1) 2–60 arbitrary units (AU): (2) 14–69 AU, (3) 61–593 AU, and (4)22–476 AU. Eight of the nine unaffected pregnancies with visible gel AChE bands had immunochemical measurements below the highest measurement for the samples with no visible AChE band (60 AU), as did five out of 20 OVWD pregnancies. Two of the OSB cases had values of 61 and 62 AU. These data indicate that the 4F19 specific monoclonal antibody to AChE is capable of distinguishing unaffected from affected pregnancies with reasonable reliability but that more work needs to be done to establish the extent of overlap between the unaffected and affected populations.     

基于磁性分离测定水样中的吲哚美辛

免疫磁性微球(SMPPA)应用于水样中吲哚美辛的分离富集,并应用高效液相色谱法对吲哚美辛进行测定。在优化的淋洗和洗脱条件下,70%甲醇和10%甲醇分别作为洗脱液和淋洗液,其回收率能达到90%。选择了湖水、河水、自来水、污水厂进口水以及医院排水为真实样品进行测定,其中湖水、河水、自来水3种真实水样中未能检测出吲哚美辛,污水厂进口水以及医院排水中吲哚美辛的含量分别为0.762、35.4 ng/m L。对这5种水进行加标回收吲哚美辛,加标回收率为92.7%~113%,RSD为1.09%~7.73%。该方法具有简便、快速、特异性等特点,能有效地分离富集水样中的目标物,对其他目标物具有广泛的实际应用价值。     

应用基于单克隆抗体的免疫传感器检测环境中的芘和苯并芘

多环芳烃是一类普遍存在的环境污染物,可以诱发癌症,对人们的健康造成严重威胁.本研究利用一种四环芳烃芘的衍生物作为半抗原,利用活泼酯法合成人工抗原免疫小鼠研制多环芳烃单克隆抗体.通过非竞争性ELISA与竞争性ELISA相结合的筛选方式得到一株单克隆抗体,命名为6A6.该抗体能特异性识别芘和苯并芘,其IC50值分别为8.1μg·L-1和6.8μg·L-1,对其他多环芳烃的交叉反应率均低于5%.在此基础上,建立了免疫传感器法对芘和苯并芘进行检测,线性范围为0.2~10.0μg·L-1.利用该方法对添加在水样中的样品进行回收,并与传统ELISA方法进行比对,结果证明传感器法检测更加快速且灵敏度更高.该方法可以用于含有芘和苯并芘的环境样品的快速筛选,可以在10 min内初步获得检测结果.     

软骨藻酸直接竞争ELISA方法的建立及优化

为建立软骨藻酸(domoic acid,DA)直接竞争ELISA方法快速检测样品中的软骨藻酸,本研究利用碳化二亚胺法将辣根过氧化物酶(HRP)标记软骨藻酸(DA),成功制得偶联物DA-HRP.在前期已制得抗软骨藻酸单克隆抗体的基础上,利用酶标抗原和单克隆抗体的特异性反应,对软骨藻酸进行定量分析.通过对封闭液、封闭时间和温育温度的优化,建立标准曲线.结果表明以1%明胶作为封闭液,在37℃下封闭1 h,加入单抗37℃温育1 h是直接竞争ELISA法的最佳反应条件,方法检出限为3.58 ng.mL-1,孔间变异系数均≤15%,加标回收率为80%～120%,检测速度较快,1.5 h内可检测出一批样品,适用于现场和批量检测,具有广泛的发展前景.     

呼吸道感染患儿血清微量元素的测定及其与抗体水平的关系

应用流动注射氢化物发生原子吸收光谱法（HGAAS）测定了呼吸道感染（RTI）患儿血清中Se和Pb,火焰原子吸收光谱法（AAS）测定Fe、Cu、Zn、Ca、Mg及Mn,光度法测定了P,以标准牛血清作质控,在最佳实验条件下,9种元素的精密度,检出限以及回收率均令人满意,根据患儿IgG,IgA,IgM补体C3及C4的测定结果,对测定方法及呼吸道感染与血清微量元素和抗体水平的关系进行了探讨,发现RTI患儿血清Se、Fe、Zn、Mn、Ca明显低于同龄正常儿童,且Ig、C3均与CK存在显著性差异。认为对RIT患儿有必要监测其血清微量元素水平。