A. Voller

Enzyme immunoassays for parasitic diseases.

It is only in recent years that serology has come to play a major role in parasitic diseases. At first complement fixation was most used but gradually more convenient and sensitive techniques such as gel precipitation (DRAPER, 1976), and agglutination methods (KAGAN, 1974) were introduced. Probably the serological test most used by parasitologists has been immunofluorescence, particularly the indirect fluorescent antibody method (KAGAN, 1974; AMBROISE-THOMAS, 1976). Certain factors must be taken into consideration when assessing the usefulness of serological methods for parasitic diseases. The major parasitoses such as malaria, schistosomiasis, trypanosomiasis, infect millions of people in poor areas of the world where technical expertise is, at present, very limited. In these areas serological methods can be of most use in establishing epidemiological indices and for monitoring disease control programmes. Tests for use in such areas should be suitable for mass screening, should be simple and cheap, and .the results should be available quickly. Under these conditions some degree of precision may have to be sacrificed in the interests of practicability. There is also a place for more sophisticated methods suitable for individual diagnosis of parasitic infections especially in the more privileged areas of the world. We feel that enzyme-immunoassays, especially the enzyme-linked immunosorbent assay, have a part to play in both the above situations.

The detection of viruses by enzyme-linked immunosorbent assay (ELISA).

The use of enzyme-linked antibodies for the detection of two morphologically different plant viruses is described. The technique is extremely sensitive, enabling assay of the viruses at concentrations as low as 10 to 100 ng/ml both in purified preparations and in crude plant extracts.

The distribution of enzyme variation in populations of Plasmodium falciparum in Africa

Isolates of Plasmodium falciparum collected from the east and west coasts of tropical Africa and also from several inland regions have been examined for electrophoretic forms of the enzymes GPI, LDH and 6PGD. Variation within and between isolates was found in the parasite forms of all three enzymes. While not disproving the existence of genetic divisions between P. falciparum populations in Africa the distribution of this variation provides no evidence for such a hypothesis.

Serum C-reactive protein levels and falciparum malaria

A microplate ELISA was developed to measure C-reactive protein (CRP) and it was used to establish the relationship between CRP levels and malaria. Highest serum CRP levels were found in African patients with high Plasmodium falciparum parastaemia. However, even African children with lower parasitaemia had higher CRP levels than others without parasitaemia. All African groups studied had CRP levels above those of a control UK group.

A comparison of isotopic and enzyme-immunoassays for tropical parasitic diseases

A comparison is made of enzyme-immunoassay and radio-immunoassay for the detection of antibody in Chagass disease, sleeping sickness, malaria, schistosomiasis ans invasive amoebiasis. Both assays were sensitive and reproductible and gave comparable results.

The development and validation of a simple antigen detection ELISA for Plasmodium falciparum malaria

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) is described for the detection of Plasmodium falciparum antigen. The test is based on an immunoglobulin (Ig) M capture monoclonal antibody on the solid phase and an IgG monoclonal antibody conjugated to peroxidase. The simple test takes about 2.5 h to complete and, because it uses whole blood with no prior treatment, it is possible to process batches of 50-100 samples simultaneously. The test is specific to P.falciparum and has a sensitivity close to that usually achieved with Giemsa-stained blood films. The reagents employed are stable at refrigerator temperatures for over 6 months, and as the test is compatible with human immunodeficiency virus and hepatitis B surface antigen ELISAs it could be suitable for blood transfusion screening.

Sero-epidemiological studies on population groups previously exposed to malaria.

Abstract Application of the indirect-fluorescent- antibody (I.F.A.) test to the study of malaria antibodies in a group of Indian and Pakistani immigrants in Bradford revealed that about 50% showed a positive response at a low titre against Plasmodium falciparum and P. vivax. There is little evidence of persistence of malaria infection in this group, but the test indicates the long duration of antibody acquired ten to fifteen years ago. In a group of potential blood-donors whose whole blood was not used for transfusion because of their possible previous exposure to malaria, only 24% were found to have a positive I.F.A. test using P. falciparum and P. malariœ antigens. A negative I.F.A. test is a good criterion of the absence of previous malaria infection.

Malarial antibodies in tropical splenomegaly syndrome in Papua New Guinea

Levels of species and class-specific malarial antibody were studied in 249 New Guineans with tropical splenomegaly syndrome (TSS) and in 87 control subjects living in the same area. Titres of IgG and IgM antibody to Plasmodium falciparum, P. vivax and P. malariae were estimated by indirect immunofluorescence. Both Ig and IgM antibody levels were higher in subjects with TSS than in controls; IgM titres were highest in those with the greatest splenic enlargement. Responses to all three species were comparable. It is concluded that there is no evidence from this study to incriminate any one species of malaria parasite in the production of tropical splenomegaly syndrome.

Role of theHLA complex in the antibody response to malaria under natural conditions

Antibodies toP. falciparum antigens and to the EB virus antigens, VCA and EBNA, were determined soon after the end of the major rainy season in 140 Africans living in 3 villages at varying altitudes in northeastern Tanzania. Also, their peripheral blood mononuclear cells were stored in liquid nitrogen and subsequently used for HLA phenotype determination of serologically determined antigens coded by genes at theA andB loci and for cell culture with mitogens (PHA, Con-A PWM) and with allogeneic cells in the mixed leukocyte reaction. A strong correlation was found between the presence of high titres of immunofluorescent antibody to falciparum antigens and the combination of A2 with AW30 in the same individuals. Individuals having one or the other of these specificities, but not both, did not have unusually high titres (P=0.0005). Individuals having the combination A2 and BW17 also tended to have higher than average antibody titres to falciparum antigens, but the difference was not statistically significant (P=0.09). The data suggests that individuals with A2 and AW30 may haveHLA-associated genes having the function ofIr genes and that these genes interact from the trans position to affect the capacity to make antibodies to malarial antigens. Thus, these genes may confer a survival advantage for individuals exposed to malaria. In the cell culture studies there were no correlations between responses and with IFA titres toP. falciparum, except for an inverse association between responses to PWM and level of IFA titre. This suggests that the B cell response to mitogens is impaired in individuals with strong responses to malarial antigens. There was no association between any of the cell culture responses and the HLA phenotypes of the cell donors.

Experimental Trypanosoma cruzi infection in rhesus monkeys—The acute phase

Abstract The course of Trypanosoma cruzi infection in 4 rhesus monkeys is described. 2 of these monkeys received Previous vaccination with a killed extract of cultural forms Plus Freunds adjuvant. While such vaccination Produced circulating antibodies in low titre and induced delayed hypersensitivity response it did not Prevent the vaccinated monkeys from developing sustained Parasitaemia. 1 of the non-vaccinated monkeys died of the T. cruzi infection. Haematological and immunological values in the course of infection are also discussed. The IgM values were not markedly raised in contrast to the situation in African trypanosomiasis.