Kumari S, Lillibridge C D, Bakeer M, Lowrie R C, Jayaraman K, Philipp M T
Department of Parasitology, Tulane Regional Primate Research Center, Tulane University Medical Center, Covington, Louisiana.
Exp Parasitol. 1994 Dec;79(4):489-505. doi: 10.1006/expr.1994.1110.
The diagnostic potential of recombinant E/S antigens of the lymphatic filaria Brugia malayi was investigated by Western blot. A cDNA expression library was constructed using B. malayi male adult worm mRNA, and E/S recombinants were identified with a rabbit antiserum raised against E/S products collected in vitro from B. malayi male and female adult worms. Two of these recombinants, Bm12 and Bm14L, were studied after subcloning the cDNA inserts in an Escherichia coli plasmid expression and purification vector, obtaining the inserts' nucleotide sequence, and purifying the expressed proteins. By homology of their deduced amino acid sequence with that of previously identified proteins, Bm12 was identified as the B. malayi gp 15/400 antigen, and Bm14 as a member of the hsp90 family of heat shock proteins. The antigenic cross-reactivity of the purified recombinant proteins was assessed with 28 serum samples from patients infected with Ascaris, Trichuris, or hookworm, and also with a few samples from patients with onchocerciasis and loiasis. For Bm12, the specificity for all of the intestinal helminthiasis together was 75%. Bm14L, on the other hand, cross-reacted with all of the ascariasis serum samples with which it was tested. Presence of antibodies cross-reactive with B. malayi was confirmed in all of these serum samples by examining their antibody reactivity with Western blots of extracts of whole B. malayi adult worms. A semiquantitative (+ or -) assessment of the sensitivity of Bm12 for antibody detection was performed using 6 serum samples from patients with chronic filariasis and 24 samples from patients with microfilaremia. All of these serum samples contained anti-Bm12 antibody (sensitivity of 100%). Finally, the ability of Bm12 to detect antibody before the onset of patency was established with a longitudinal collection of serum samples obtained from 2 African green vervets (Cercopithecus aethiops) and 3 rhesus macaques (Macaca mulatta), all of which were infected with B. malayi. Anti-Bm12 antibodies were detectable in all animals between 4 and 11 weeks before patency.
通过蛋白质印迹法研究了马来布鲁线虫重组排泄分泌（E/S）抗原的诊断潜力。利用马来布鲁线虫雄性成虫mRNA构建了一个cDNA表达文库，并用针对从马来布鲁线虫雌雄成虫体外收集的E/S产物制备的兔抗血清鉴定E/S重组体。将这些重组体中的两个，即Bm12和Bm14L，在将cDNA插入片段亚克隆到大肠杆菌质粒表达和纯化载体中、获得插入片段的核苷酸序列并纯化表达的蛋白质后进行研究。通过其推导的氨基酸序列与先前鉴定的蛋白质的氨基酸序列的同源性，Bm12被鉴定为马来布鲁线虫gp 15/400抗原，Bm14被鉴定为热休克蛋白hsp90家族的成员。用来自感染蛔虫、鞭虫或钩虫患者的28份血清样本以及一些来自盘尾丝虫病和罗阿丝虫病患者的样本评估了纯化重组蛋白的抗原交叉反应性。对于Bm12，对所有肠道蠕虫病的总体特异性为75%。另一方面，Bm14L与所有检测的蛔虫病血清样本发生交叉反应。通过检测这些血清样本与马来布鲁线虫成虫全虫提取物的蛋白质印迹的抗体反应性，证实了所有这些血清样本中存在与马来布鲁线虫交叉反应的抗体。使用来自慢性丝虫病患者的6份血清样本和来自微丝蚴血症患者的24份血清样本对Bm12检测抗体的敏感性进行了半定量（+或-）评估。所有这些血清样本均含有抗Bm12抗体（敏感性为100%）。最后，通过纵向收集从2只非洲绿猴（猕猴）和3只恒河猴（恒河猴）获得的血清样本，确定了Bm12在虫体出现之前检测抗体的能力，所有这些动物均感染了马来布鲁线虫。在虫体出现前4至11周，在所有动物中均可检测到抗Bm12抗体。
