Rodero M, Jiménez A, Cuéllar C
Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense, Madrid, España.
Mem Inst Oswaldo Cruz. 2002 Mar;97(2):247-52. doi: 10.1590/s0074-02762002000200018.
In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of (1/4)00 and 1 microg/ml of antigenic concentration.
为提高人体异尖线虫病诊断技术的特异性和敏感性，已开发出一种亲和色谱法，用于从简单异尖线虫第三期幼虫（L3）中纯化种特异性抗原。用简单异尖线虫或猪蛔虫抗原免疫新西兰兔，或用犬弓首蛔虫感染性虫卵接种新西兰兔。通过蛋白A-琼脂糖CL-4B珠柱分离IgG特异性抗体。将抗简单异尖线虫和抗猪蛔虫的IgG偶联到溴化氰活化的琼脂糖4B上。为纯化幼虫简单异尖线虫抗原，将其加载到抗简单异尖线虫柱中，洗脱结合的抗原。为消除负责交叉反应的表位，将简单异尖线虫特异性蛋白加载到抗猪蛔虫柱中。为证明分离蛋白的特异性，进行了聚丙烯酰胺凝胶电泳免疫化学分析。此外，我们通过酶联免疫吸附测定（ELISA）研究了对所用不同简单异尖线虫抗原制剂的不同反应，观察它们区分兔体内产生的不同抗血清（抗简单异尖线虫、抗猪蛔虫、抗犬弓首蛔虫）的能力。使用幼虫简单异尖线虫粗提物（CE）抗原时，与抗犬弓首蛔虫抗血清的区分能力良好。当将幼虫简单异尖线虫CE抗原加载到与用简单异尖线虫CE抗原免疫的兔IgG偶联的溴化氰活化琼脂糖4B上时，其区分简单异尖线虫和猪蛔虫的能力得到提高，对犬弓首蛔虫的区分能力也有所增加。使用加载到与用成年猪蛔虫CE抗原免疫的兔IgG偶联的溴化氰活化琼脂糖4B上的幼虫简单异尖线虫CE抗原可获得最佳结果。当我们比较不同的血清稀释度和抗原浓度时，选择了（1/4）00的工作血清稀释度和1微克/毫升的抗原浓度。
