Olave Ana M, Mesa Jairo A, Botero Jorge H, Patiño Edwin B, García Gisela M, Alzate Juan F
Grupo de Parasitología, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia.
Biomedica. 2016 Mar 3;36(1):39-51. doi: 10.7705/biomedica.v36i1.2617.
Toxocara canis is a pathogenic nematode of canines which can be accidentally transmitted to humans. Although serology is the most important diagnostic tool for this zoonosis, diagnostic kits use crude excretion/secretion antigens, most of them being glycoproteins which are not species-specific and may cross-react with antibodies generated against other parasites.
To produce the rTES-30 recombinant antigen of Toxocara canis and evaluate it in the immunodiagnosis of toxocariasis.
The gene that codes for TES-30 was cloned in the expression vector pET28a (+) using single-stranded oligonucleotides united by PCR. The protein rTES-30 was purified by Ni2+ affinity chromotography. Seroreactivity of rTES-30 was evaluated by immunoblot. Given that there is no gold standard test, the behaviour of the antigen was compared with the method that is routinely used to immunodiagnose toxocariasis, i.e., the conventional ELISA technique using excretion/secretion antigens.
The rTES-30 was produced from an Escherichia coli LB culture which yielded 2.25 mg/L of the antigen with a purity of 95%. The results obtained showed 73% (46/63) concordance of reactivity between the rTES-30 immunoblot and the conventional ELISA, and 100% concordance with the nonreactive sera (21). Nineteen of the 21 sera positive for other parasitoses reacted with ELISA, while only seven of these were positive with the rTES-30 immunoblot. Concordance between the ELISA and the immunoblot was moderate (kappa coefficient: 0.575; 95% CI: 0.41- 0.74).
The data presented show the potential of the rTES-30 inmunoblot for confirmation of possible ELISA positives, not only in epidemiological studies, but also as a candidate for the development of diagnostic tests for ocular toxocariasis in Colombia.
犬弓首线虫是犬类的一种致病性线虫，可意外传播给人类。尽管血清学是这种人畜共患病最重要的诊断工具，但诊断试剂盒使用的是粗排泄/分泌抗原，其中大多数是糖蛋白，它们并非物种特异性的，可能会与针对其他寄生虫产生的抗体发生交叉反应。
制备犬弓首线虫的rTES - 30重组抗原，并在弓首线虫病的免疫诊断中对其进行评估。
使用通过PCR连接的单链寡核苷酸，将编码TES - 30的基因克隆到表达载体pET28a(+)中。通过Ni2+亲和层析纯化rTES - 30蛋白。通过免疫印迹评估rTES - 30的血清反应性。鉴于不存在金标准检测方法，将该抗原的表现与常规用于弓首线虫病免疫诊断的方法（即使用排泄/分泌抗原的传统ELISA技术）进行比较。
rTES - 30由大肠杆菌LB培养物产生，该培养物产生浓度为2.25mg/L、纯度为95%的抗原。所得结果显示，rTES - 30免疫印迹与传统ELISA之间的反应性一致性为73%（46/63），与无反应血清的一致性为100%（21份）。21份其他寄生虫病阳性血清中有19份与ELISA反应，而其中只有7份与rTES - 30免疫印迹呈阳性反应。ELISA与免疫印迹之间的一致性为中等（kappa系数：0.575；95%可信区间：0.41 - 0.74）。
所呈现的数据表明，rTES - 30免疫印迹不仅在流行病学研究中，而且作为哥伦比亚眼弓首线虫病诊断试验开发的候选方法，在确认可能的ELISA阳性结果方面具有潜力。
