Monoclonal antibodies to third stage larva lf gnathostoma spinigerum : antibody specificities and antigen characterization

Abstract

Monoclonal antibodies (McAbs) to third stage larva of Gnathostoma spingerum (L(,3)G) were produced by fusing BALB/c immune spleen cells with myeloma cells strain x63.Ag8.653. The characteristics of these clones were tested by using ELISA, immunoblot, radioimmunoprecipitation (RIP), and immunofluorescence technique. Forty-one clones of hybrid cells were obtained form 14 mother clones in this fusion. Thee clones secreted antibodies reactiong with somatic antigen of L(,3)G (Som.L(,3)G) when tested by ELISA. A sample of one clone developed from each mother clone was tested for its antibody specificity by ELISA against antigen of L(,3)G, Angiostrongylus cantonensis, Brugia jpahangi, Opisthorchis viverrini, Clonorchis sinensis, Faciola gigantica. Eleven McAbs gave IgG1/k isotype antibodies specific to both Som.L(,3)G and excretory/recretory antigens of L(,3)G (ES.L(,3)G). There was only one clone that secreted IgM/K antibody cross-reacted with antigen of B. pahangi. lymphatic filarial worm of cat, two other clones secreted IgM/k antibodies reacted with bovine serum albumin. McAbs 21.3 and 31.4, the L(,3)G specific antibodies were selected for further characterization along with the cross-reactive one, McAb 1F2. By immunoblot technique, McAbs 21.3 and 31.4 were found to react with the same group of protein molecules of Som.L(,3)G having MW ranging from 97-34 KD and 53-21 kD in ES.L3G, while McAb 1F2 only reacted with ES.L(,3)G at MW ranging from 49-38 kD. The antigenic epitopes represented on protein molecules of Som.L3G recognized by McAbs 21.3 and 31.4 were found to be non-carbohydrate in nature, when tested by periodate oxidation combining with immunoblot technique. When RIP technique was used, mcAbs 21.3 and 31.4 were found to react with antigenic epitopes on 38 kD protein molecule of Som.L(,3)G and antigenic epitopes on 49, 45, 33, 29, 16, and 14 kD protein molecules of ES.L(,3)G. The McAb 1F2, on the other hand was found to react with epitopes on 49 and 45 kD protein molecules of ES.L(,3)G, but did not react with Som.L(,3)G. These three McAbs were used to localize the tissue possessed the specific antigenic epitopes on cryosections of L(,3)G by immunofluorescence technique. It was demonstrated that McAb 21.3 reacted with epitopes on intestinal cells, while McAb 31.4 reacted with epitopes on both the intestinal cells and muscle walls. McAb 1F2 on the other hand was found to react with basal lamina component. However, when the test was done with living L(,3)G, it was clearly shown that these mcAbs did not react with the cuticular surface of the parasite. The results of the present study were discussed and tried to apply by using McAb 31.4 to detect for ES.L(,3)G in cerebrospinal fluid (CSF) of twenty patients suffering from cerebral disturbance using biotin-streptavidin ELISA which has the sensitivity at 125 ng/ml. The ES.L3G could be detected in only one patients CSF which had low free form antibody level against ES.L3G in CSF.