[Experimental study on hybridoma cells agglutination test for diagnosis of schistosomiasis japonica in mice].

OBJECTIVE: To detect the infection of Schistosoma japonicum in mice with a novel test based on agglutination of hybridoma cells and to study the mechanism of the hybridoma cells agglutination. METHODS: The procedure was developed with a murine cell line H226 producing a monoclonal antibody specific to schistosome 31/32 kDa antigen and sera collected from mice infected with different numbers (10, 30, 50) of S. japonicum cercariae in different period. Immunofluorescent test was carried out with the hybridoma cells and schistosome-infected sera. RESULTS: The circulating antigen was detected by the test as early as 2 weeks after a heavy infection and all mice showed positive results in the test by 5 weeks after infection. The titers of antigen rose along with the time post infection, and the titers of sera from heavy infection were statistically higher than that from the mice receiving a lower number of cercariae. Specific yellowish green fluorescence appeared on the membrane of the hybridoma cells; no signal was detected inside. CONCLUSION: Hybridoma cell agglutination test (HCAT) may become useful to diagnose schistosomiasis.

Hybridoma cell agglutination as a novel test to detect circulating antigen of Schistosoma japonicum.

We developed a serodiagnostic test which is based on the agglutination of hybridoma cells. In the presence of specific antigen, agglutination of the fixed and stained cells occurs and can be visualized in analogy to traditional erythrocyte agglutination. The procedures were developed with a murine cell line producing a monoclonal antibody against a schistosome gut protein and sera of patients and mice infected with Schistosoma japonicum. This test is capable of detecting circulating antigen during pre-patency in mice infected with 50 cercariae. Its sensitivity was high with acute schistosomiasis japonica (97%, n = 32) and moderate with chronic cases (75%, n = 57). No positive reactions were obtained with healthy persons (n = 78) or patients infected with other parasites (Chlonorchis sinensis, n = 20; Paragonimus westermani, n = 20; Plasmodium vivax, n = 10) or suffering from lupus erythomatodus (n = 5) or mononucleosis (n = 10).

Detection of circulating antigens and parasite specific antibodies in filariasis.

In Peninsular Malaysia, only Wuchereria bancrofti and Brugia malayi are reported to cause human filariasis. Brugia pahangi infects many of the same animal hosts as the zoonotically transmitted subperiodic B. malayi. There is a well-recognized need for improved diagnostic techniques for lymphatic filariasis. Parasite antigen detection is a promising new approach, and it will probably prove to be more sensitive and specific than clinical, microscopic and antibody-based serological methods. We recently generated monoclonal antibodies (MAb XC3) from in vitro culture products of adult B. pahangi (B.p. IVP). Filarial antigenemia was quantitated in various hosts including the sera from 6 Malaysian Aborigines with acute lymphatic filariasis. In hosts infected with brugian filariasis and dirofilariasis, antigenemia was scored ranging from 90 ng/ml to 960 ng/ml. None of the control animal and human sera had antigenemia above 90 ng/ml. In addition, MAb XC3 and B.p. IVP were applied in several seroepidemiological surveys among household cats in Kuala Selangor in order to correlate information gathered for future studies of possible cases of human infection. Out of the 81 cats surveyed, 10 (12.35%) and 5 (6.17%) were parasitologically positive for B. pahangi and B. malayi, respectively. However, 21 (25.92%) were antigenemia positive when serologically investigated with MAb XC3. Antifilarial antibodies to B.p. IVP by direct ELISA showed very high cross-reactivity with non-filarial gut worm infections. 16 (19.75%) cats had reciprocal titers ranging from 320 to 2,560. Only 1 (1.23%) cat from this group was antigenemic.

In vivo selection of populations of Plasmodium chabaudi chabaudi AS resistant to a monoclonal antibody that reacts with the precursor to the major merozoite surface antigen.

Mice bearing a hybridoma secreting a monoclonal antibody (MAb), MAb-3, which significantly delays the onset of a Plasmodium chabaudi chabaudi AS, but not P. chabaudi chabaudi CB, challenge parasitemia in a passive transfer assay and which is specific for the precursor to the major merozoite surface antigen (PMMSA) of P. chabaudi chabaudi AS, were challenged intravenously with 10(3) P. chabaudi chabaudi AS-parasitized erythrocytes. The resultant parasitemia was very similar to that in normal mice except that initially the parasitemia was sometimes slightly delayed. Parasites derived from cryopreserved stabilates isolated from MAb-3 hybridoma mice with an unmodified parasitemia, or with a delayed parasitemia, were found to have lost their susceptibility to MAb-3 in the passive transfer assay. A number of anti-PMMSA MAb were used to immunoprecipitate lysates of parasite populations isolated directly from hybridoma-bearing mice. In some instances and with certain of the MAb, immunoprecipitation patterns were modified, but other isolates were not detectably different when compared with unselected P. chabaudi chabaudi AS parasites. Using a panel of MAb reacting with the PMMSA of P. chabaudi chabaudi AS, immunoprecipitation patterns of parasites derived from cryopreserved stabilates isolated from hybridoma-bearing mice were determined at 2-h intervals through the appropriate part of the parasite maturation cycle. In these derived populations, resistance to MAb-3 was not associated with a change in the immunoprecipitation reaction with the MAb used. These results are discussed in the context of current knowledge of genotypic and phenotypic antigenic diversity of malaria parasites and other protozoa.

Expression of protozoan antigens by hybrids between Leishmania mexicana and myeloma SP2/0.

Hybrid cells were obtained between Leishmania mexicana promastigotes and mouse myeloma SP2/0 cells, and examined for expression of leishmanial antigens. A ratio of 1:10 of myeloma to T. cruzi cells was unsuccessful because of outgrowth of non-fused cells. With a ratio of 2:1 four waves of multiplication of 'chimeric' cells were observed over 45 days. The death of the hybrids after this period is explained by segregation of DNA and loss of chromosome material. Hybrid cells gave a positive reaction with antibodies in the sera from patients infected with Leishmania, as demonstrated by indirect immunofluorescence. Conversely, promastigote forms of Leishmania gave a negative reaction with the same antibodies, which recognize surface antigens of the amastigote stage of Leishmania. It is possible therefore that amastigote stage antigens are expressed on the surface of the 'chimeric' hybrid as a result of transformation of promastigotes following hybridization.

Antigens of the erythrocytes stages of the human malaria parasite Plasmodium falciparum detected by monoclonal antibodies.

A range of 22 mouse anti-P. falciparum monoclonal antibodies have been characterized by indirect immunofluorescence and immunoprecipitation. On the basis of these studies, 5 groups of antibodies and 6 classes of antigen were defined. Group I antibodies give, bright, uniform, generalised staining of all blood stages including gametocytes. Three of these antibodies precipitate a metabolically labelled molecule(s) of 35 kDa. One precipitates a 50 kDa antigen. Group II antibodies, which give strong localised immunofluorescence in merozoites, and a weak diffuse pattern in earlier stages, precipitate biosynthetically labelled molecules of 160 kDa. Group III antibodies react with all asexual stages. With merozoites they produce intense staining around the perimeter, both in fixed and unfixed preparations. They precipitate biosynthetic molecules of 190 kDa. Group IV antibodies are identical to Group III except they are stage restricted to schizonts and merozoites. They also precipitate 190 kDa antigens. These, however, in contrast to group III, are readily accessible to 125I-lactoperoxidase labelling. One antibody also precipitates a set of smaller peptides. Finally, Group V antibodies produce very bright ill-defined staining of pigment-containing parasites, as well as of inclusions in the red cell. They precipitate a series of molecules of 160, 60 and 35 kDa which are readily accessible to 125I. The 160 kDa molecule is also labelled by [35S]methionine. These results are discussed in the context of the development of a malaria vaccine and immunodiagnostic tests.