Immunoassays for tropical parasitic infections

Abstract

Introduction Over the last decade there has been an explosion of interest in and development of immunodiagnostic tests for infectious diseases. The technical developments are most easily considered in terms of reagents and of methods. The tremendous advances in biotechnology have meant that the biggest changes have been in the reagents, namely, antibodies and antigens used. Monoclonal antibodies have been raised to virtually every antigen of all the parasitic protozoa. These antibodies are highly specific and can provide the basis of much improved tests for antigen detection. Monoclonal antibodies are now used routinely as the raw material in immunofluorescent tests (e.g., to demonstrate Entamoeba) and in enzymelinked immunosorbent assays (ELISAS) (e.g., for malaria antigen detection); they have the great advantage that large amounts of defined, standardized reagents can be produced. Monoclonal antibody-based ELISAS or dot-blot systems have proved most valuable for the demonstration of protozoa1 parasites, or their antigens, in their vertebrate hosts, vectors and the environment. One result of the recent emphasis on vaccine development has been a much improved understanding of the make-up of parasites, and this is having a significant impact on immunodiagnostics. The analysis of the structure of the major antigens has led to the identification of those specific antigens best suited for use in tests to detect the host’s antibodv response. Such antigens can be nrenared on a large scale by-peptide synthesrs or by recombinant technoloav and can markedlv imnrove the snecificitv of tests for Gtibody (e.g. ELI& for *Trypanosom> cruzi ahtibody). The developments in methods have been less dramatic than those in reagents in recent years, and consist mainly of improvement and consolidation of existing methods rather than truly innovative approaches. Those techniques which employ a ‘labelled’ reagent dominate the field at present. One of these, immunofluorescence (or fluorescent antibody) was for long the test of choice but it has now largely been replaced by ELISA which, using enzymes as labels, is more suitable for large scale use and which yields objective results. ELISA has the added advantage of being useful for both antigen and antibody detection. The various dot-blot tests are essentially similar but use antigens on paper or plastic supports to detect antibody which is then visualized using antiglobulins labelled with enzymes, dyes or colloidal gold. These dotblot methods are more convenient, especially as ‘dipstick’ techniques, even though they may be less suitable for large scale screening applications. The PATH@ HIV dipstick is an example of a dot system using colloidal gold and is easy and cheap to use, and gives rapid reliable results (KERCKHOVEN et al., 1991). It is seen by some as the model for other test systems of the future. Simple agglutination tests are attractive propositions and would be ideal for use in the tropics if they could be made reliable. Those dependent on antigen/antibody coated ervthrocytes are -invariably troublesome and give substantial numbers of false positive reactions in the tronics. This is one reason why ihey have gone out of fashion for hepatitis B surface antigen (HBsAg) testing. However, a new agglutination test for human immunodeficiency virus (HIV) based on antigen coupled to a monoclonal antibody against human red blood cells is more suitable for tropical use, since it uses the patient’s own red blood cells as the indicator (RYLATT et al., 1990). Again, this test format could be extended to other infections. Other agglutination tests using parasites or fragments have also come into prominence for trypanosomiasis and leishmaniasis and are discussed below. The real ‘state of the art’ immunoassays are the pregnancy tests currently available. These have reached a new level of user-friendliness and are based on devices incorporating monoclonal antibody-coated beads which, in the presence of human chorionic gonadotrophin, will produce a coloured reaction product. The devices require no measurement of sample or timing, and medical or laboratory training is not required to use them. Unfortunately their cost atpresent puts them beyond the reach of all but the wealthiest nations. Similar devices have been introduced for Chlamydia and for HIV. Until recently, most immune tests were done on serum. This is a rather less attractive proposition nowadays because of the hazards associated with blood collection and attempts have been made to use alternative noninvasive sampling. Urine is the well established choice for pregnancy tests and has been used for the detection of antigens in schistosomiasis and Chagas disease. Possibly of more general application is antibodv detection in saliva and urine. With properly designed capture ELISAS these give results as crood as serum in an HIV antibodv test ~MORTIMER &-PARRY! 1991) and, presumably, they could be used for detectmg other infections.