ELISAs for parasitologists: or lies, damned lies and ELISAs

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ParasitologyToday.vol. 9. no. 6. 1993 z

ELISAs for Parasitologists: or Lies, D a m n e d Lies and ELISAs P. Venkatesan and D. Wakelin The introduction in 197 I, by Engvall and Perlmannl, of enzyme labels in immunoassays represented a significant technical advance. Their enzyme-linked immunosorbent assay (ELISA) proved to be as sensitive as radioimmunoassays but safer to use. Since then, ELISAs have been widely used in the assay of anti bodies and antigens. In this article, Pradhib Venkatesan and Derek Wakelin focus on some of the problems associ ated with ELISAs when applied to para sitic infections, which can modify antibody responses, by immunosuppression or polyclonal activation. The article also considers optimization, which is an essential step in the establishment of an ELISA.

Alistair Voller has pioneered the application of the enzyme-linked immunosorbent assay (ELBA) to numerous infections 2. The ELISA has now become a standard procedure in parasitology. In the research laboratory, ELISAs are widely used in studying serological responses to parasitic infections. In the clinical setting, they have been developed for diagnostic purposes, either to demonstrate the appearance of antibody or to detect parasite antigens3,4. In the clinical setting, there are limitations to the application of the ELISA. Current diagnostic techniques may be adequate, rendering an ELISA unnecessary; and in the developing world, the necessary equipment and reagents are often unavailable. Nonetheless, there are certain situations in which an ELISA is particularly useful. Parasitological diagnosis is often dependent on visualizing the parasite in blood, excreta or tissue biopsies. However, the presence of the parasite (eg. Bancroftian filariasis) in the bloodstream may be intermittent and transient, and in low-grade, chronic gastrointestinal infections (eg. strongy Ioidiasis) parasites are frequently absent from faecal specimens. In such situations, serological diagnosis by ELISA has advantages5,6. Furthermore, ELISA can be used for large-scale batch testing in epidemiological studies6. The ELISA has an important role in the laboratory and in the field. As with

any investigative tool, researchers must be aware of the scope and limitations of the technique used. Figure I shows the various components involved in a standard indirect ELISA. A solid phase, usually a plastic microtitre plate, is coated with antigen onto which is placed serum, an anti-immunoglobulin enzyme conjugate and an enzyme substrate for incubation. These components will be discussed below. Solid Phase

A solid support surface can be pro vided by beads, tubes or microtitre plates. The latter is by far the most convenient, as centrifugation steps and the use of large sample volumes are avoided. As a standard plate contains 96 wells, analysis of a large number of samples is possible. The microtitre plates may be made of different materials (eg. polystyrene and polyvinyl chloride) or be specially treated (eg. coated with poly-[ lysine). Polyvinyl chloride has the greater capacity to bind proteins. This is advantageous in binding antigens, but disadvantageous in increasing the non-specific binding of antibodies to vacant plastic sites. The main problem encountered with plates is that of batch-to-batch variability, and sometimes within-batch variability7&

trate

Anti-immunoglobulin enzyme conjugate

Support surface Fig. I. The components of a standard indirect ELISA.

Thus, the same specimens on different plates may give different optical-density readings. The only way to control for this is to have appropriate standards on every plate. Antigen

The nature of the plastic protein interactions involved in binding antigen to plates is not completely understood, although it is thought that hydrophobic forces play the major part. In coating plates with antigen, the aim is for uniformit 7 within and between plates. Antigen binding varies depending on the nature of the antigen, and in an heterogeneous mixture some components will adsorb more strongly than others 9. in general, water-soluble proteins are more likely to bind. Greater uniformity is achieved with less heterogeneous preparations or, ideally, singleantigen preparations. If coating is carried out at 4°C, the cooler, outer wells may bind less antigen than the inner wells. This 'edge effect' occurs when equilibrium has not been reached, and applies to all the stages of an ELISA '°, Furthermore, if plates are stacked, the lower and upper plates may differ from the middle plates. Therefore, one must be wary of stacking when incubation steps involve changes in temperature from room temperature to 4°C or 37°C. In coating plates, maximal adsorption, retained antigenicity and minimal desorption are desirable aims. Antigen adsorption increases with concentration, temperature and time of incubation, up to a plateau t ~.q2 The height of the plateau can depend on the pH of the coating buffer. For instance, polysaccharide antigens such as the pneumococcal capsular antigens require a buffer at pH 3.0 for optimal adsorption r3, in contrast to the commonly used carbonate bicarbonate buffer at pH 9.6. Adsorption can also be enhanced by crosslinking proteins~! This has been achieved without affecting the affinity of antibody. In some cases, excess antigen, beyond the con© 1993, Flsevier Science Publishers lid, (UK)

Parasitology Today, vol. 9, no. 6, 1993

centration required to achieve the plateau, can lead to overcrowding and concealment of epitopes ~s. Consequently, antibody binding diminishes. This can also happen when binding alters the conformation of antigenic epitopes, reducing antibody recognition 16. Desorption depends on the nature of the plastic plate, the antigen and the number of washing steps. Studies using radiolabelled antigen have demonstrated that up to 30% of antigen initially adsorbed can be lost during the course of an ELISA 17. Conformational change and desorption are not practical problems in many ELISAs. When they are, crosslinking of antigen may be an answer. However, for most ELISAs, optimization of antigen coating is confined to identifying the best antigen concentration and coating buffer. This is done using titrations of antigen in buffers at pHs of, for instance, 5.0, 7.4 and 9.6 (Ref. 18). Conditions that give the highest plateau are chosen. For many proteins, the plateau is reached at concentrations above I i~g ml t. The usual way of finding the optimal antigen concentration is to use immune serum and proceed with a standard ELISA. If the serum used has a high affinity, it will bind to much-lower concentrations of antigen than a low-affinity serum. Thus, in optimizing with a high-affinity serum, one could inadvertently choose an antigen concentration that is not detected by lower-affinity sera 19. The sensitivity of the ELISA is thereby reduced. Thus, it is preferable either to optimize with low-affinity sera or to use a method that is independent of antibody affinity, such as the peroxidase saturation technique 20. In this technique, peroxidase is incubated on plates coated with antigen. Any vacant plastic sites are filled by the enzyme and its presence can be detected by the addition of substrate. Over a range of antigen concentrations, minimal peroxidase activity corresponds to maximal antigen coating.

Binding of Antibodies in Serum The binding of antibodies in immune serum has two components: (I) there is nonspecific binding to exposed sites on the plastic microtitre plate or to the antigen itself] and (2) there is specific binding to epitopes on the antigens. Nonspecific binding should be minimized and specific binding should be maximized. Nonspeofic binding. There are two approaches to minimizing nonspecific

229

binding: 'prevention' and 'cure'. The 'cure' approach is to wash thoroughly. However, specifically bound antibody, as well as the nonspeciflc, can be washed away. 'Prevention' can be achieved by blocking vacant plastic sites with protein. Various proteins have been tried and compared for their suitability as blocking agents 2t. Skimmed milk and casein are better blockers than bovine serum albumin (BSA). These proteins not only exhibit protein-plastic interactions, but also protein antibody interactions. This property can be exploited by diluting serum in phosphate-buffered saline (PBS) containing skimmed milk or BSA. Protein bound to antibody then reduces the nonspecific binding of the antibody. However, there are two problems with protein blockers. (I) Human serum can contain antibodies to BSA and casein, causing false-positive reactions22. (2) Antibody-protein-plastic interactions may indirectly increase nonspecific binding, particularly over long incubation times ~8.An alternative approach to minimizing nonspecific reactions is to use the detergent Tween 20 in PBS23 as this 'wets' hydrophilic proteins such as immunoglobulins (Igs). When PBSTween is used for serum dilutions, prorein-blocking agents may not offer any additional benefit 24. Therefore, it can be argued that the widely used blocking step can be abandoned or reserved for situations when Tween 20 has to be omitted (eg. because the antigen is detergent-sensitive due to a lipid-containing epitope). Each ELISA system must be treated individually, and Tween 20 should be compared with blocking agents for optimizing nonspecific backgrounds. Specific binding. The specific reaction of antigen and antibody proceeds to an equilibrium and the time taken to reach that equilibrium depends on temperature and antibody concentration. When specific antibody is present in high titre, equilibrium may be reached within two hours, but if it is present in low titre it may take longer. To maximize specific binding, incubation with serum should not be terminated before equilibrium is reached. It is useful to perform a kinetic study of antibody binding to determine the optimal duration of incubation. While higher temperatures increase the rate of reaction, they can also enhance nonspecific binding and antigen desorption.

Optimizing specific and nonspeciflc reactions. Measures taken to decrease nonspecific binding may also decrease specific binding. On the other hand,

measures taken to increase specific binding may also increase nonspecific binding. Finding the optimal balance depends on being able to distinguish specific and nonspecific components, and preferentially limiting the latter. One method used for distinguishing specific and nonspecific binding is to use positive and negative sera. Conditions are chosen to minimize the optical density obtained with negative sera and maximize the difference in optical densities between positive and negative sera. Having studied and optimized washing, blocking reagents or Tween 20, one has to decide whether to analyse samples at a single dilution or with a titration to an endpoint.

Assays, Titrations and Choices

Single-dilution assays. It is helpful to know the end-point titre of a positive sample. The overall optical density is the sum of specific and nonspecific binding represented by the two curves shown in Fig. 2. There is a critical dilution on this curve beyond which there is only nonspecific binding and proximal to which there are both specific and nonspecific components. This dilution is the true end-point titre of specific antibodies. It may be identified by concomitant titrations of positive and negative sera. However, positive and negative sera can differ in important respects. In some parasitic infections there is a polyclonal activation of B cells resulting in increased production of nonspecific antibodies (the classical examples being malaria and trypanosomiasis2S). In laboratory animals, antibody levels change with age and rise with bacterial and viral infections on entering animal houses. Therefore, positive serum, compared with negative serum, may contain more antibody, and this may lead to enhanced nonspecific binding. The titration curve for immune serum may then be higher than non-immune serum (Fig. 3). (The converse may apply if the parasitic infection is associated with suppression of antibody production2k) Thus, in such cases, assays performed at dilutions beyond the true end-point will demonstrate a difference between immune and non-immune serum that is due entirely to nonspecific binding (Fig. 4). For this reason, in animal experiments, it may be appropriate to use age-matched control serum rather than pre-immune serum.

Parasitology Today, vat. 9, no. 6, 1993

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Fig. 4. (Left) A rise in optical density due entirely to nonspecif~ binding as the serum samples were diluted beyond the true end-point. Fig. 5. (Right) The sensitivity of anti-immunoglobulin enzyme conjugates. When different dilutions of conjugate are used to demonstrate specific binding by immune serum, very high dilutions fail to detect all specific antibodies. As the dilution is lowered the sensitivity is increased and more specific antibodies are detected until the true end-point is reached. Beyond this, lowering the dilution changes the gradient of the titration curve and increases non-specific binding of the conjugate. (Dilutions: closed circles, I : 500; open squares, I : 1000; closed squares, I : 2000; open triangles, I : 4000; dosed triangles, I : 8000; open circles, naive serum.)

Knowing the true end-point, an assay dilution can be chosen on the specific section of the titration curve. This end-point sets the higher limit of dilution. Statistical considerations influence the lower limit. Let us say that the end-point dilution is I :500. Assay at I :100 will then only permit detection of a fivefold increase in antibody binding, while assay at I: 50 permits detection of a tenfold increase. Furthermore, the dilution chosen determines sensitivity: for instance, with a dilution of I : 100, all titres below this will be counted as negative. While statistics and sensitivity favour a low dilu-

tion, limits are set by sample economy, nonspecific reactions and possible prozone reactions (a prozone being a paradoxical fall in optical density with decreasing dilution of serum). Having defined a range of possible dilutions the final consideration is to choose a dilution on a steep or linear part of a titration curve as this increases the sensitivity for changes in antibody binding. End-point titres. The end-point may be defined as the titre at which the positive sample dilution equilibrates to a negative sample. Alternatively, it may be defined as the dilution which

reaches a pre-set optical density, chosen arbitrarily, perhaps as a percentage of the optical density of a standard sample. The choice. H o w does one choose between single-dilution assays and endpoint titrations? These methods have been compared 3.27. Titration to an endpoint involves more plates, more reagents and more time. As the endpoint is determined at low levels of antibody binding, this method is very dependent on the sensitivity of the assay. Single-dilution assays are more economical with regard to time and reagents, but optical densities are sub-

Parasitology Today, vol. 9, no. 6, 1993

ject to plate-to-plate and day-to-day variability. A compromise is to use single dilutions, but relate the optical densities to a standard curve derived from reference serum. A standard titration curve on every plate takes into account plate-to-plate and day-to-day variability.

Anti-immunoglobulin Enzyme Conjugates and Substrates In optimizing the use of conjugates, specificity and sensitivity must be considered. The conjugate provided by suppliers should be specific for the stated isotype and should not crossreact with other isotypes. Lack of crossreactivity is very likely to be the case with the major isotypes but problems may be encountered with isotype subclasses (eg. IgG). That conjugate can also bind nonspecifically to vacant plastic sites and to the antigen layer is indicated by control wells to which serum has not been added. The sensitivity of the conjugate in detecting antibody bound to antigen depends on the affinity and the concentration of conjugate used. The concentration must be sufficient to detect all specific antibody binding and identify the true end-point titre (Fig. 5). The concentration chosen is determined by reagent economy, obtaining a steep titration curve and minimizing nonspecific background. A variable to be aware of is the stoichiometric relationship of conjugate binding to antibody. Using radiolabelled antibodies, it is possible to show that over a wide range of concentrations there is a linear relationship between antibody concentration and binding to antigen 28. However, over the same range of concentrations, addition of conjugate and substrate results in a sigmoid curve for optical densities (aDs). This can be partly explained by a change in the stoichiometric ratio of conjugate to antibody. With IgM, for example, there is a total often heavy chains in one molecule. For a conjugate antibody directed against the heavy chain the number of antibodies binding to an IgM molecule could, therefore, vary from one to ten. Such variation in the ratio of conjugate to antibody does occur in practice, and the O D observed does not always give a linear guide to antibody concentration 29. A change in the conjugate:antibody ratio is more likely over long incubation periods and with higher concentrations of conjugate. Variation

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in stoichiometry may be one of many contributory factors to overall day-today variability. The conjugated enzyme and its substrate are chosen for sensitivity and convenience. Ideally, the enzyme should rapidly convert several substrate molecules to produce a strong colour signal with a single, narrow absorption peak. The stronger the signal for each enzyme molecule the more sensitive is the assay. Enzymes and substrates have been compared 3°,31. The two main enzymes used are horse-radish peroxidase (HRP) and alkaline phosphatase (ALP). The latter is slightly simpler to use, but it is claimed that HRP with the substrate 3,3',5,5'-tetramethylbenzidine (TMB) is the more sensitive32.

Results: Interpretation and Expression Enzyme-linked immunosorbent assays provide a very sensitive method for detecting specific antibodies. Qualitative information on the presence or absence of such antibodies is easy to obtain by statistical comparison of optical densities from test and control sera. However, obtaining quantitative information is not so straightforward. Indirect ELISAs measure antibody binding and not antibody levels. Antibody binding is a function of both antibody affinity and concentration. A serum sample with high-affnity antibody present in low concentration may give the same binding and optical density as a serum sample with lowaffinity antibody present in high concentration. Therefore, a standard indirect ELISA does not give absolute quantitative information. Even endpoint titres are only titres of antibody binding. Comparisons of antibody binding can be made. However, a doubling in optical density does not necessarily mean a doubling in antibody binding. One can only make this conclusion if there is a linear relationship between optical density and dilution, which is not always the case. When the relationship is non-linear, quantitative changes in binding can only be deduced from end-point titres or by reference to a standard curve.

Caveats The main pitfall in dealing with results is overinterpreting data and drawing quantitative conclusions when

only qualitative conclusions are possible. It is important for authors to quote a coefificient of variation (CV) so that readers can have some idea of the variability of results. The CV is calculated using standard samples from standard deviation of a d s divided by the mean of aDs, and expressed as a percentage. One can get caught out by unexpected, false-positive reactions. These may occur because of crossreactivity 33-3s or because of unpredictable nonspecific reactions 36.37. For instance, Haralabidis has described crossreactions on ELISA between Echinococcus

granulosus, Fasciola hepatica, Giardia lamblia, Leishmania donovani, Taenia saginata, Toxoplasma gondii and Trichinella spiralis 33. To check for unpredictable nonspecific reactions, one can include controls in which antigen is added to the serum diluent. This antigen will inhibit the specific binding of antibody to antigen on the plate, but will not affect any nonspeciflc reaction.

Conclusions Despite potential pitfalls and problems, the ELISA remains an excellent and valuable assay. However, it is important to be aware of its limitations and not overinterpret results, tt is also important to tailor each ELISA to suit the system. One cannot presume that the method used in one system will be the optimal method in another. Only a simple indirect ELISA has been discussed here but many of the points apply to more sophisticated applications of the ELISA technique.

Acknowledgements The authors gratefully acknowledge the helpful comments on this manuscript given by Herbert Sewell, Faroukh Shakib, Rhodri Jones and Karen Robinson. Pradhib Venkatesan is a Wellcome Trust Medical Graduate Research Fellow.

References

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Global Infectious Diseases: Prevention, Control and Eradication

found the chapters on arboviruses and on hepatitis particularly interesting. The way modem molecular techniques were applied to identify hepatitis C virus (HCV) is no less impressive than the strategies used to identify HIV. The analogy does not end there. While writing this review the French Minister of Health (Bernard Kushner) announced that, in France, between S00000 and two million people are infected with HCV. He invited everybody that had received a blood transfusion in the past 15 years to come forward for testing. Hepatitis C virus causes the majority of post-transfusion N A N B (non-A/non-B) hepatitis and good serodiagnosis is now only possible due to modem molecular techniques. The ability to detect HCV in infected blood, helps prevent transmission of N A N B hepatitis by transfusion. Today, no review of global diseases would be complete without a chapter on AIDS. The one written by June E. Osbom is a good synopsis of what was understood a couple of years ago (an inevitable time lag where books are concemed). The depressing point is that, in spite of AIDS being a preventable disease, new people are still being infected. I will come back to this point at the end. As stated in my introduction, most people (including myself) will read this book to learn about diseases outside their particular field of interest. My field of interest for the past 12 years has been malaria, so it was with a certain degree of expertise that I read the chapter by Carter L. Diggs on 'Prospects for Control of Malaria in the Twenty-first Century'. This is one of the largest chapters which probably reflects both the importance of the disease and

the number of scientists working on it! When dealing with generalities and the political/economic aspects of malaria control, Diggs gives a knowledgeable overview, as one would expect from a Director of the malaria programme for the US Agency for International Development (AID). However, his review of vaccine development is overly restricted; for instance, the Patarroyo blood-stage vaccine is hardly discussed, and transmission-blocking vaccines are not mentioned at all. The review by Eric A. Ottesen on filariasis I read with interest, due to my lack of knowledge about this important family of parasites (that affects I00 million people). I was surprised when he stated that one of the future challengers will be the development of stage (L 2, L3 and L4)-specific c D N A libraries for differential antibody screening, without mentioning genomic D N A expression libraries. This is particularly relevent, because many of the problems of in vitro culture Ottesen frequently invoked for filariasis, are also true of plasmodia hepatic stages. Differential antibody screening of genomic expression libraries has been used to isolate malaria hepatic stage antigen genes and it seems reasonable to suppose they would also be useful in filariasis. One of the last chapters concerns cysticercosis, and this excellent review starts with the statement that the most important aspect of the disease is that it is preventable. As June Osbom mentioned, AIDS is also a preventable disease and yet every day more and more people are infected. These examples clearly indicate to us that the real problems posed by global infectious diseases, are problems of

edited by David H. Walker, Springer-Verlag, 1992. DM 148.00 (x + 234 pages) ISBN 3 211 82329 8 The reasons that led me to agree to review this book are probably the same as would lead someone to buy the book., ie, a desire to be informed about the different important worldhealth problems, without the time to keep up with the detailed scientific literature. I enjoyed the collection of reviews and can recommend it to potential buyers. The recommendation however, is not without reserve and these reservations (both general and specific) I will outline below. My general criticisms are restricted to just two points, and the first conceres more a question of style. In spite of the use of 'Global' in the title of the book, the different world-health problems are described from a predominantly American point of view. A nonAmerican public may find this a little irritating, and I think an opportunity was lost by not having a 'global' view of a 'global' problem. The second point is the absence of a chapter on leishmaniasis in a review of 'Global Infectious Diseases'. There are -350 million people at risk to leishmaniasis with more than 12 million people infected. Leishmaniasis is surely a much more global problem, than, for example, Lyme's disease. The above points aside, the book is easy to read with contributions from leading workers in each of the fields discussed. As a molecular biologist, I

Pradhib Venkatesan and Derek Wakelin are at the Department of Life Science, University o f Nottingham, Nottingham, UK NG7 2RD.