228 Parasitology Today. 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 con- siders optimization, which is an essential step in the establishment of an ELISA.

Alistair Voller has pioneered the appli- cation of the enzyme-linked immuno- sorbent assay (ELBA) to numerous infections 2. The ELISA has now become a standard procedure in para- sitology. In the research laboratory, ELISAs are widely used in studying serological responses to parasitic infec- tions. In the clinical setting, they have been developed for diagnostic pur- poses, either to demonstrate the appearance of antibody or to detect parasite antigens 3,4.

In the clinical setting, there are limi- tations to the application of the ELISA. Current diagnostic techniques may be adequate, rendering an ELISA unnecess- ary; 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 visual- izing 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 situ- ations, serological diagnosis by ELISA has advantages 5,6. Furthermore, ELISA can be used for large-scale batch test- ing in epidemiological studies 6.

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 sub- strate for incubation. These com- ponents 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 ma- terials (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 variability 7&

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.

Ant igen

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 uni- formit 7 within and between plates. Antigen binding varies depending on the nature of the antigen, and in an heterogeneous mixture some com- ponents will adsorb more strongly than others 9. in general, water-soluble pro- teins are more likely to bind. Greater uniformity is achieved with less hetero- geneous preparations or, ideally, single- antigen 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 stack- ing when incubation steps involve changes in temperature from room temperature to 4°C or 37°C.

In coating plates, maximal adsorp- tion, retained antigenicity and minimal desorption are desirable aims. Antigen adsorption increases with concen- tration, temperature and time of incu- bation, 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 com- monly used carbonate bicarbonate buffer at pH 9.6. Adsorption can also be enhanced by crosslinking proteins~! This has been achieved without affect- ing the affinity of antibody. In some cases, excess antigen, beyond the con-

© 1993, Flsevier Science Publishers l id, (UK)

Parasitology Today, vol. 9, no. 6, 1993 229

centration required to achieve the plateau, can lead to overcrowding and concealment of epitopes ~s. Conse- quently, antibody binding diminishes. This can also happen when binding alters the conformation of antigenic epitopes, reducing antibody recog- nition 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 anti- gen initially adsorbed can be lost during the course of an ELISA 17.

Conformational change and desorp- tion are not practical problems in many ELISAs. When they are, crosslinking of antigen may be an answer. However, for most ELISAs, optimization of anti- gen coating is confined to identifying the best antigen concentration and coating buffer. This is done using ti- trations 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 concentra- tion is to use immune serum and pro- ceed 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 anti- gen 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 tech- nique 20. In this technique, peroxidase is incubated on plates coated with anti- gen. 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 concentra- tions, minimal peroxidase activity cor- responds 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 mini- mized and specific binding should be maximized.

Nonspeofic binding. There are two approaches to minimizing nonspecific

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 suit- ability as blocking agents 2t. Skimmed milk and casein are better blockers than bovine serum albumin (BSA). These proteins not only exhibit pro- tein-plastic interactions, but also pro- tein antibody interactions. This prop- erty can be exploited by diluting serum in phosphate-buffered saline (PBS) containing skimmed milk or BSA. Pro- tein 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 reactions 22. (2) Antibody-protein-plastic interactions may indirectly increase nonspecific binding, particularly over long incuba- tion times ~8. An alternative approach to minimizing nonspecific reactions is to use the detergent Tween 20 in PBS 23 as this 'wets' hydrophilic proteins such as immunoglobulins (Igs). When PBS- Tween is used for serum dilutions, pro- rein-blocking agents may not offer any additional benefit 24. Therefore, it can be argued that the widely used block- ing 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-con- taining epitope). Each ELISA system must be treated individually, and Tween 20 should be compared with blocking agents for optimizing non- specific 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 concen- tration. When specific antibody is pre- sent 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, incuba- tion with serum should not be termi- nated 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 non- specific 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. Con- ditions 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 end- point.

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 di- lution 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 lab- oratory 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 associ- ated with suppression of antibody pro- duction2k) 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 non- specific binding (Fig. 4). For this reason, in animal experiments, it may be appropriate to use age-matched con- trol serum rather than pre-immune serum.

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

- L o g d i l u t i o n - L o g d i l u t i o n

Fig. 2. (Left) The observed optical density (open squares) is the sum of specif~: (closed circles) and nonspecific (open circles) components. As specific antibodies represent a fraction of total antibodies, specifc binding disappears, on serial dilution of serum, before nonspecific binding does. The point of disappearance is the true end-point

Fig. 3. (Right) Titration curves for serum samples with quantitatively different amounts of antibody. As nonspecif,: binding rises (from open circles to closed circles) with the increased quantity of antibody, the titration curves are shifted upwards. Differences in optical density between the serum samples (open and dosed squares) may, therefore, be due entirely to the nonspecif~ component.

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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 influ- ence the lower limit. Let us say that the end-point dilution is I :500. Assay at I :100 will then only permit detec- tion of a fivefold increase in antibody binding, while assay at I : 50 permits detection of a tenfold increase. Fur- thermore, the dilution chosen deter- mines sensitivity: for instance, with a dilution of I : 100, all titres below this will be counted as negative. While stat- istics and sensitivity favour a low dilu-

tion, limits are set by sample economy, nonspecific reactions and possible pro- zone 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 per- centage of the optical density of a stan- dard sample.

The choice. How does one choose between single-dilution assays and end- point titrations? These methods have been compared 3.27. Titration to an end- point involves more plates, more reagents and more time. As the end- point 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 231

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 titra- tion 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 con- sidered. The conjugate provided by suppliers should be specific for the stated isotype and should not cross- react with other isotypes. Lack of crossreactivity is very likely to be the case with the major isotypes but prob- lems may be encountered with iso- type subclasses (eg. IgG). That con- jugate 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 con- centration of conjugate used. The con- centration 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 non- specific 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 of ten 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 OD observed does not always give a linear guide to antibody concentra- tion 29. A change in the conjugate:anti- body ratio is more likely over long incubation periods and with higher concentrations of conjugate. Variation

in stoichiometry may be one of many contributory factors to overall day-to- day variability.

The conjugated enzyme and its sub- strate 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 peroxi- dase (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 sensitive 32.

Results: Interpretation and Expression

Enzyme-linked immunosorbent assays provide a very sensitive method for detecting specific antibodies. Quali- tative 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. Anti- body binding is a function of both anti- body affinity and concentration. A serum sample with high-affnity anti- body present in low concentration may give the same binding and optical density as a serum sample with low- affinity antibody present in high con- centration. Therefore, a standard indi- rect ELISA does not give absolute quantitative information. Even end- point 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 re- lationship is non-linear, quantitative changes in binding can only be de- duced 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 poss- ible. 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 calcu- lated 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 un- expected, false-positive reactions. These may occur because of crossreac- tivity 33-3s or because of unpredictable nonspecific reactions 36.37. For instance, Haralabidis has described crossreac- tions on ELISA between Echinococcus granulosus, Fasciola hepatica, Giardia lamblia, Leishmania donovani, Taenia saginata, Toxoplasma gondii and Tri- chinella spiralis 33. To check for unpredict- able nonspecific reactions, one can include controls in which antigen is added to the serum diluent. This anti- gen will inhibit the specific binding of antibody to antigen on the plate, but will not affect any nonspeciflc reaction.

Conclusions

Despite potential pitfalls and prob- lems, 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 dis- cussed here but many of the points apply to more sophisticated appli- cations 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.

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9 Kenny, G.E. and Dunsmoor, C.L. (1983)J. Gin. Microbial. 17, 655 665

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19 M~kela, O. and P~terfy, F. (1983) Eur. J.

Global Infectious Diseases: Prevention, Control and

Eradication

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 world- health 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 con- ceres more a question of style. In spite of the use of 'Global' in the title of the book, the different world-health prob- lems are described from a predomi- nantly American point of view. A non- American 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 leishmani- asis in a review of 'Global Infectious Diseases'. There are -350 million people at risk to leishmaniasis with more than 12 million people infected. Leishmani- asis is surely a much more global prob- lem, 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

Immunol. 13, 81S 819 20 ML~noz, C. et al. (1986)J. Immunol. Methods

94, 137 144 21 Vogt, R.F. et al. (1987)J. Immunol. Methods

101,43 50 22 Shetty, N.P., Raj, I.$. and Macaden, R.8. (I 990)

J. Clin. Pathol. 43,950 952 23 Gardas, A. and Lewartowska, A. (1988)

J. Immunol. Methods 106, 251 255 24 Mohammad, K. and Esen, A. (1989)

J. Immunol. Methods I 17, 141 145 25 Greenwood, B.M. (I 974) Lancet i, 435~436 26 Monroy, F.G. and Enriquez, FJ. (1992) Poro

sitology Today 8, 49 54 27 Malvano, R. et al. (1982)J. Immunol. Methods

48, 51 60 28 Koertge, T.E. and Butler, J.E. (1985) J.

Immunol. Methods 83, 283 299 29 Pruslin, F.H. et ol. (199 I) J. Immunol. Methods

137, 27 35 30 AI-Kaissi, E. and Mostratos, A. (1983)

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) an- nounced that, in France, between S00000 and two million people are infected with HCV. He invited every- body that had received a blood trans- fusion 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 poss- ible due to modem molecular tech- niques. The ability to detect HCV in infected blood, helps prevent transmis- sion 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 pre- ventable 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

J. Immunol. Methods 58, 127 132 31 Porstmann, B. et al. (1985) J. Immunol

Methods 79, 27 37 32 Madersbacher, S. and Berger, P. (1991)

J. Immunol. Methods 138, 121 124 33 Haralabidis, S.T.H (1984) Ann. Trap. Meal

Parasitol. 78, 295 300 34 Pritchard, D.I. et al. (1991) Trans. R. Sac Trap.

Med Hyg. 85, 51 1-514 35 Timothy, L.M. et al. (1992) Int. J. Parasitol 22,

1143 1149 36 Williams, J.E. and Robinson, D.M. (1982)

Trans. R. Sac. Trap. Meal Hyg. 76, 280 28 I 37 Laurent, M.S. et al. (1990)J. Immunol. Methods

133, 145 146

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

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 Devel- opment (AID). However, his review of vaccine development is overly re- stricted; 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 chal- lengers will be the development of stage (L 2, L 3 and L4)-specific c D N A libraries for differential antibody screen- ing, without mentioning genomic D N A expression libraries. This is particularly relevent, because many of the prob- lems of in vitro culture Ottesen fre- quently invoked for filariasis, are also true of plasmodia hepatic stages. Dif- ferential 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 pre- ventable 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