Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.     

Management of nucleus and IOL drop

Background: Thirty six cases of lenticular nucleus drop following phacoemulsification and 43 cases of posterior dislocation of intraocular lens (IOL) inclusive of two paediatric cases were managed by a modified vitrectomy procedure without using perfluorocarbon liquid (PFCL).     

用电化学检测器的高效液相色谱法测定血清及尿中速尿的含量

本文报告了用电化学检测器的高效液相色谱法测定血清及尿中速尿含量的方法。样品予处理方法:血清用乙腈除蛋白,尿用蒸馏水稀释50倍。采用作者合成的FD-Val-OH作为内标物。色谱条件为:反相柱,以含35%乙醇的5mmol/L四丁基铵水溶液为流动相(pH7.50),流速1.0ml/min;用电化学检测器,检测电压0.90V:速尿及内标物的保留时间分别为10和15min。通过计算速尿对内标物的峰高比求得速尿含量。血清及尿中的最低检测浓度分别为16和9ng/ml。标准曲线在0.25～5ng/μl(血清)、0.5～10ng/μl(尿)的浓度范围内呈线性关系。血清及尿中回收率分别为100.5%和100.6%。变异系数在4.6%以下。     

Immunoblotting characterization of neutrophil antigenic targets in autoimmune neutropenia

We characterized neutrophil autoantigens using an immunoblotting technique with antibodies obtained from patients with autoimmune neutropenia. These results were correlated with serologic characterization of the antibodies, using indirect immunofluorescence and leukoagglutination. Of the 17 sera immunoblotted, 16 showed discrete bands in the molecular weight range of 30 to 112. Three patients with Felty's syndrome reacted with an antigenic target of 80 to 84 Kd molecular mass, a finding not seen in any of the other patients studied. By serologic testing, none of the autoimmune sera showed serologic specificity for any known neutrophil-specific alloantigen. Using an anti-NA-1 serum, we identified antigenic targets at 40, 50, and 101 Kd in both NA-1-positive and NA-1-negative neutrophils. Ten of 17 autoimmune sera showed reactivity in this corresponding range. These studies demonstrate that immunoblotting may be used to identify antigenic targets in autoimmune neutropenia and may suggest a specificity of these antibodies not definable by serologic techniques. Correlation of immunoblot reactivity with disease states associated with immune neutropenia may be useful in the study of the pathogenesis of the different forms of autoimmune neutropenia.     

心肌坏死标记物在检测心肌坏死中的应用与地位

目的:评估各种心肌坏死标记物对判断心肌坏死的价值。资料来源:应用计算机检索LWW全文英文数据库1994-01/2006-08与心肌坏死标记物相关的文献,检索词“Myocardial damage,myocardiolysis,marker”,并限定文章语言种类为“English”。资料选择:对资料进行初审,选对于临床有意义的心肌坏死物进行较详细的总结。选择临床研究的文章,无论有无对照组、是否为随机对照文献均纳入;排除观察对象为动物的基础研究和综述类文献。资料提炼:对收集到的文献进一步查找全文,凡是对于心肌坏死检测快速且可靠的心肌坏死物优先选择,研究内容相似的,以近3年且发表在较权威杂志者优先,最后纳入30篇关于心肌坏死标记物的文章进行综述。资料综合:急性血栓形成及其随后的心肌损伤、坏死是急性心肌梗死的最主要特征,准确、快速、可靠地检测血栓形成和心肌坏死标志物对内科医生诊断急性心肌梗死极具价值。研究表明缺血修饰白蛋白、心肌型脂肪酸结合蛋白、髓过氧化物酶、CK-MB和心肌肌钙蛋白对急性心肌梗死的诊断各具特点,而联合检测更有助于早期、准确诊断急性心肌梗死,为尽早抢救急性心肌梗死创造时机。结论:缺血修饰白蛋白、心肌型脂肪酸结合蛋白、髓过氧化物酶、CK-MB和心肌肌钙蛋白是较敏感的心肌坏死标记物,联合应用诊断价值更大。