Review of critical factors affecting analytical characteristics of serological and molecular assays.

Analytical characteristics of diagnostic tests, encompassing estimates of repeatability, analytical specificity (ASp) and analytical sensitivity (ASe), are determined during Stage 1 of the OIE Assay Validation Pathway. Repeatability (an estimate of assay precision and robustness), ASp (measuring only what an assay is intended to measure) and ASe (synonymous with the lower limit of detection) are fundamental parameters that determine future test performance. Importantly, these parameters provide the basis for deciding whether a prototype assay progresses to the next stage of the OIE Assay Validation Pathway (determination of diagnostic characteristics) or is withdrawn in favour of alternate tests with better analytical performance characteristics. Implicit in the successful development and validation of any assay is a sound understanding of the target pathogen, the disease pathogenesis in susceptible hosts, the fundamental technical principles that underliey each test system, and its intended use. Factors that affect analytical characteristics of diagnostic assays are numerous and may vary according to each assay type. Using, as examples, development of an enzyme-linked immunosorbent assay for detection of antibodies to capripoxviruses, and the comparative assessment of three quantitative real-time polymerase chain reactions for detection of African swine fever virus DNA, the main factors affecting analytical characteristics of serological and molecular assays are considered. As reviewed within, comprehensive and well-designed experiments are required to develop and optimise assays with favourable analytical characteristics. The underlying principles are broadly applicable to all assay types and, when conducted with appropriate rigour, provide the foundations for high-quality diagnostic tests that are fit for their intended purpose(s).

Les caractéristiques de performance analytique des tests diagnostiques, qui recouvrent l'estimation de la répétabilité, de la spécificité analytique (SpA) et de la sensibilité analytique (SeA) d'un test sont déterminées lors de l'étape 1 du processus de l'OIE relatif à la validation des essais. La répétabilité (une estimation de la précision et de la robustesse de l'essai), la SpA (qui mesure uniquement ce que l'essai est destiné à mesurer) et la SeA (synonyme de limite inférieure de détection) sont des paramètres essentiels qui déterminent les futures performances du test. Il est important de noter que ces paramètres apportent les éléments essentiels pour décider si l'essai peut passer à l'étape suivante du processus de validation de l'OIE (détermination des caractéristiques diagnostiques) ou s'il doit céder la place à des tests alternatifs dotés de meilleures caractéristiques de performance analytique. Pour réussir la mise au point et la validation d'un essai, certaines conditions préalables doivent être réunies : bien connaître l'agent pathogène cible et la pathogenèse de la maladie chez les réservoirs sensibles, ainsi que les grands principes techniques sous-jacents à chaque système de test et l'emploi prévu du test. Les facteurs affectant les caractéristiques analytiques d'un essai diagnostique sont nombreux et varient suivant le type d'essai dont il s'agit. À partir d'exemples portant sur une épreuve immuno-enzymatique mise au point pour la détection des anticorps dirigés contre les capripoxvirus et sur l'évaluation comparative de trois techniques d'amplification en chaîne par polymérase quantitative en temps réel pour la détection de l'ADN viral de la peste porcine africaine, les auteurs mettent en exergue les principaux facteurs qui peuvent altérer les caractéristiques analytiques des essais sérologiques et moléculaires. Il ressort de cette évaluation que des expérimentations complètes et bien conçues sont nécessaires pour mettre au point et optimiser des essais possédant les caractéristiques analytiques souhaitées. En général, les principes sous-jacents sont applicables à tous les types d'essai, et s'ils sont appliqués de manière rigoureuse, ils fournissent la garantie de disposer de tests diagnostiques de qualité élevée et aptes à l'emploi ou aux emplois prévus.

La primera etapa del proceso de validación de ensayos de la OIE es aquella en que se determinan las características analíticas de una prueba de diagnóstico, o dicho de otro modo, en que se calculan los valores de repetibilidad (estimación de la precisión y robustez del ensayo), especificidad analítica (es decir, el hecho de que el ensayo mida únicamente lo que está destinado a medir) y sensibilidad analítica (sinónimo referido al límite inferior de detección), que son tres parámetros fundamentales para determinar el futuro rendimiento de una prueba. Un aspecto importante es que estos parámetros sientan las bases a partir de las cuales decidir si un prototipo de ensayo debe pasar a la siguiente etapa del proceso de validación de ensayos de la OIE (determinación de las características de diagnóstico) o si vale más retirarlo en beneficio de otras pruebas que presenten mejores características de rendimiento analítico. Un factor implícito en el éxito de todo proceso de desarrollo y validación de ensayos es un sólido conocimiento del patógeno en cuestión, la patogénesis de la enfermedad en los anfitriones sensibles, los principios técnicos fundamentales en que reposa cada sistema de ensayo y sus usos previstos. Los numerosos factores que influyen en las características analíticas de un ensayo de diagnóstico difieren en función del tipo de ensayo. Utilizando como ejemplo el desarrollo de un ensayo inmunoenzimático de detección de anticuerpos contra capripoxvirus y la evaluación comparativa de tres PCR cuantitativas en tiempo real para detectar ADN del virus de la peste porcina africana, los autores pasan revista a los principales factores que determinan las características analíticas de los ensayos serológicos y moleculares. Como explican, para desarrollar y optimizar ensayos que presenten características analíticas favorables se requieren experimentos completos y bien concebidos. Los principios subyacentes son válidos en general para todo tipo de ensayos y, cuando se aplican con el debido rigor, sientan las bases para obtener pruebas de diagnóstico de gran calidad y adaptadas a la(s) finalidad(es) prevista(s).

Introduction - Validation of tests for OIE-listed diseases as fit-for-purpose in a world of evolving diagnostic technologies.

The World Organisation for Animal Health (OIE) has made leading contributions to the discipline of test validation science by providing standards and guidelines that inform the test validation process in terrestrial and aquatic animals. The OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, and the Manual of Diagnostic Tests for Aquatic Animals describe the test validation pathway in the context of fitness for purpose, elaborate on the importance of diagnostic sensitivity (DSe) and specificity (DSp) as measures of test accuracy, and designate additional factors (e.g. test cost, laboratory throughput capacity and rapidity of test results) that influence choices of a single test over others or the inclusion of a new test in a diagnostic process that includes multiple tests. This paper provides examples of each of the six main testing purposes listed in the Terrestrial Manual and describes additional metrics such as ruggedness and robustness that should be included in the validation of point-of-care tests. Challenges associated with new diagnostic technologies and platforms are described. Validated tests with estimates of DSe and DSp are needed to measure confidence in test results for OIE-listed diseases, to facilitate risk assessments related to animal movement, to estimate true prevalence, and for certification of disease freedom and use in epidemiological (risk factor) studies.

L'Organisation mondiale de la santé animale (OIE) a apporté d'importantes contributions dans le domaine de la validation des tests en élaborant des normes et des lignes directrices qui informent sur le processus de validation des tests chez les animaux terrestres et aquatiques. Le Manuel des tests de diagnostic et des vaccins pour les animaux terrestres et le Manuel des tests de diagnostic pour les animaux aquatiques de l'OIE décrivent le processus de validation des tests dans le contexte de leur aptitude à l'emploi, expliquent l'importance de la sensibilité (DSe) et de la spécificité (DSp) diagnostiques pour mesurer l'exactitude des tests, et désignent d'autres facteurs (ex. coût des tests, capacité de traitement des laboratoires et rapidité d'obtention des résultats des tests) qui influencent le choix d'un test par rapport à un autre ou l'inclusion d'un nouveau test dans un processus de diagnostic composé de multiples tests. Le présent article fournit des exemples pour chacun des six principaux objectifs définis pour les tests figurant dans le Manuel terrestre et décrit des mesures supplémentaires, telle la robustesse (aussi bien interne qu'externe), qu'il conviendrait d'inclure dans la validation des tests au point d'intervention. Il aborde également les défis soulevés par les nouvelles technologies et plateformes de diagnostic. Des tests validés accompagnés d'estimations de la DSe et de la DSp sont nécessaires pour mesurer la fiabilité des résultats des tests pour les maladies listées par l'OIE, faciliter les évaluations des risques associés aux mouvements des animaux, estimer le véritable taux de prévalence et certifier l'absence de maladies ; ils sont également indispensables pour les études (des facteurs de risque) épidémiologiques.

La Organización Mundial de Sanidad Animal (OIE), con su labor de elaboración de normas y directrices que fundamentan el proceso de validación de pruebas para enfermedades de los animales terrestres y acuáticos, ha hecho aportaciones punteras a la disciplina científica que se ocupa de la validación de pruebas. En su Manual de las Pruebas de Diagnóstico y de las Vacunas para los Animales Terrestres y su Manual de las Pruebas de Diagnóstico para los Animales Acuáticos, la OIE describe el procedimiento de validación de pruebas en clave de idoneidad para determinados propósitos, ahonda en la importancia de la sensibilidad y la especificidad diagnósticas (DSe y DSp) como medidas de la exactitud de una prueba y señala otros factores (como el costo de la prueba, la productividad del laboratorio o la rapidez de los resultados) que también influyen en la elección de una determinada prueba por delante de otras o en la inclusión de una nueva prueba en un proceso de diagnóstico que entraña el uso de varias. Los autores ofrecen ejemplos de cada uno de los seis principales propósitos con las que puede utilizarse una prueba, según vienen enunciados en el Manual Terrestre, y describen otros parámetros que es preciso tener en cuenta a la hora de validar pruebas practicadas en el punto de consulta, como la robustez o también la solidez (ruggedness en inglés; llamada a veces «robustez interlaboratorios¼). También describen las dificultades ligadas a nuevas tecnologías y plataformas de diagnóstico. Se necesitan pruebas validadas y acompañadas de un cálculo de la DSe y la DSp para fines tan diversos e importantes como medir la confianza que merecen los resultados de pruebas para enfermedades inscritas en las listas de la OIE, facilitar la evaluación del riesgo ligado al desplazamiento de animales, estimar la prevalencia real, certificar la ausencia de enfermedad o realizar estudios epidemiológicos (factores de riesgo).

Development and validation of a 3ABC antibody ELISA in Australia for foot and mouth disease.

OBJECTIVE: To measure the diagnostic performance of an Australian-developed ELISA for the detection of antibodies against the non-structural proteins (NSP) 3ABC of the foot and mouth disease (FMD) virus. DESIGN: Test development and validation study. METHODS: The diagnostic specificity was determined using 2535 sera from naïve animals and 1112 sera from vaccinated animals. Diagnostic sensitivity was calculated from the data for 995 sera from experimentally and field-infected animals from FMD-endemic countries in South East Asia. A commercial ELISA detecting antibodies against FMD virus NSP was used as the reference test to establish relative sensitivity and specificity. Bayesian latent class analysis was performed to corroborate results. The diagnostic window and rate of detection were determined at different times using sera from cattle, sheep and pigs before and after infection, and after vaccination and subsequent infection. Repeatability and reproducibility data were established. RESULTS: At 35% test cut-off, the 3ABC ELISA had an overall diagnostic sensitivity of 91.5% and diagnostic specificity of 96.4%. The diagnostic sensitivity in vaccinated and subsequently infected cattle was 68.4% and diagnostic specificity in vaccinated cattle was 98.0%. CONCLUSIONS: The 3ABC ELISA identified field and experimentally infected animals, as well as vaccinated and subsequently infected animals. Diagnostic sensitivity and specificity estimates for other FMD NSP tests are comparable with the results obtained in this study. This NSP ELISA was found to be 'fit for purpose' as a screening assay at the herd level to detect viral infection and also to substantiate absence of infection.

Validation of a PCR-oligochromatography test for detection of Trypanozoon parasites in a multicenter collaborative trial.

A PCR-oligochromatography test for diagnosis of human and animal trypanosomiasis was evaluated through a multicenter ring trial with six laboratories testing a set of 21 blinded samples, resulting in qualitative data (positive or negative). Results showed an intralaboratory repeatability (accordance) of 88.7% (credible interval [CI], 84.4 to 92.5%) and an interlaboratory repeatability (concordance) of 88.1% (CI, 84.3 to 92.3%).

Aspects of kit validation for tests used for the diagnosis and surveillance of livestock diseases: producer and end-user responsibilities.

The Joint Food and Agriculture Organization/International Atomic Energy Agency (IAEA) Division of Nuclear Techniques in Food and Agriculture, based at the IAEA in Vienna, Austria, has extensive experience in helping to develop and validate assays and has provided strong support in developing World Organisation for Animal Health (OIE) norms. This paper will focus on enzyme-linked immunosorbent assay and polymerase chain reaction as the major technologies exploited in diagnosis and surveillance. Problems involving the terminology and factors in kit production, supply and validation are examined, in particular emphasising the importance of robustness and ruggedness of tests. The authors discuss the responsibilities of the various stakeholders (producers, distributors, users, and national/international organisations) in achieving quality controlled data to solve diagnostic and surveillance problems. The roles of internal quality control (internal proficiency testing) and external quality assurance (external proficiency testing) as well as aids to solving problems with kits are examined.

Development and field validation of an avidin-biotin enzyme-linked immunosorbent assay kit for bovine brucellosis.

The avidin-biotin enzyme-linked immunosorbent assay (A-B ELISA), for use in surveillance for bovine brucellosis in India was developed and calibrated using the indirect brucellosis ELISA kit of the International Atomic Energy Agency (IAEA) as a reference. The reagents used in the A-B ELISA were as follows: the smooth lipopolysaccharide of Brucella abortus strain 99 (antigen); biotinylated anti-bovine immunoglobulin G (detection antibody); avidin-horseradish peroxidase (conjugate); and O-phenylenediamine dihydrochloride (chromogen). The test results were interpreted using the IAEA software EDI version 2.1.1, which was modified for use in the A-B ELISA. The cut-off percentage positivity value was established using 500 brucellosis-positive and 500 brucellosis-negative serum samples, confirmed with reference to the sample data using the indirect ELISA kit. The overall specificity of A-B ELISA was 98.8% and overall sensitivity was 98.2%. Field validation of the A-B ELISA kit was undertaken in six laboratories in India. Screening of 7,040 cattle and 678 buffalo serum samples from 12 states revealed serological evidence of brucellosis in 8.7% of cattle and 10.2% of buffalo. This kit proved to be robust and performed with a similar sensitivity and specificity to the indirect ELISA. The kit can be supplied at a lower cost than current commercial ELISA kits.

Effect of gamma-irradiation on serum samples on the diagnostic performance of ELISA methods for the detection of trypanosomal antibodies.

The study investigated the effect of gamma-irradiation on bovine serum samples on the ability of enzyme-linked immunosorbent assay (ELISA) methods to detect trypanosomal antibodies. The serum samples were analysed using two standardised indirect ELISA systems. Higher measurement values were observed for most gamma-irradiated antibody positive and negative test samples. Using cut-off points, determined from the analysis of a non-irradiated trypanosomal antibody-negative population, the gamma-irradiated sera data showed that there was an increased risk of misclassifying samples as false positive or cross-reactive due to increased analytical sensitivity and decreased analytical specificity. The intraplate precision and agreement between tested and expected values of measurements were not altered throughout. The impact on the assays' diagnostic performance was estimated by analysing diagnostic sensitivity, diagnostic specificity and related parameters. The data demonstrated that although there was a bias of higher measurement values after gamma-irradiation, this could be compensated after readjustment of the cut-off points to obtain best separation of antibody-positive and -negative samples. Thus, for each assay, no significant difference of the diagnostic proficiency was found before and after gamma-irradiation. The practical implications are discussed of a serum sterilisation procedure using (60)Co gamma-rays for routine sample testing, assay validation and trypanosomosis monitoring and tsetse-fly control and eradication programmes.

Charting methods to monitor the operational performance of ELISA method for the detection of antibodies against trypanosomes.

Four indirect enzyme-linked immunosorbent assays (ELISAs) for the detection of antibody against trypanosomes using antigen-precoated plates (Trypanosoma congolense and T. vivax) were used in 15 veterinary diagnostic laboratories in Africa and Europe. The study provided data allowing an evaluation of charting methods with respect to the operational performance of each ELISA. Data from standardised internal quality control (IQC) samples were plotted on charts and used as the assay performance indicators with reference to expected upper and lower control limits. Based on unprocessed (optical density) and normalised absorbance values (calculated as a percentage positivity of a control), dispersion of values from the expected data range was estimated plotting the location and deviation of the values. In addition, assay precision was estimated plotting the distribution of coefficients of variation<10% of the IQCs. Binding ratios of controls were calculated to estimate the assay proficiency with respect to the accuracy of assessing that the IQC samples tested positive or negative in the test proper. The graphical analysis of dispersion of absorbance values in combination with assay precision and proficiency criteria was considered fully satisfactory to evaluate the operational performance of the ELISAs and provided useful decision criteria for plate acceptance and rejection. The establishment of standardised and transparent IQC data charting methods for the indirect ELISAs provided an increased measure of confidence to national laboratories with respect to their reports on disease occurrence. Moreover, the relative assay performances between all laboratories were examined using summary data charts with reference to the performance criteria described. The IQC data were also examined using modified Youden plot analysis demonstrating that indirect ELISA methods can be successfully applied at diagnostic laboratories in the tropics for monitoring trypanosomosis control programmes.

Evaluation of antigen-coating procedures of enzyme-linked immunosorbent assay method for detection of trypanosomal antibodies.

Research was undertaken to improve the antigen-coating step of indirect enzyme-linked immunosorbent assay (ELISA) method through the use of polystyrene 96-well plates precoated with antigenically stabile crude trypanosomal antigens. The plates were precoated with antigens, air dried and sealed before being packed in plastic bags with silica gel desiccant packets. Such plates stored at +4 and +37 degrees C provided an assay performance, which was superior to that of plates freshly coated with antigens from a frozen stock. Antigen-precoated plates consistently proved stable after storage up to +50 degrees C for at least 1 year. The accuracy of the assay was not affected, i.e. trypanosomal antibody-positive sera were clearly discriminated from trypanosomal antibody-negative negative sera. In contrast, lyophilized trypanosomal antigens lacked stability on storage at +37 degrees C for longer than 1 month. It was concluded that the routine use of antigen precoated polystyrene plates for the enzyme immunoassay technique will contribute to improved assay robustness at an acceptable diagnostic proficiency. The modified coating procedure will also provide an improved quality assurance and standardization procedure for the assay, which is required to allow the reliable detection of trypanosomal antibodies and comparison of data from different laboratories.

Detection of Trypanosoma congolense antibodies with indirect ELISAs using antigen-precoated microtitre plates.

The study reports the performance of four indirect enzyme-linked immunosorbent assays (ELISAs) for antibody (AB) detection using microtitre plates which were precoated with native or heat/detergent denatured antigens (AGs) from Trypanosoma congolense (T.c.) and T. vivax (T.v.), and stored for between 1 to 206 days at +37 degrees C. Bovine serum samples were obtained by sequential bleeding of 3-months old T.c.-infected bulls and their uninfected cohorts, as well as by a single bleeding of uninfected adult cattle. The first day of AB detection, and observations on samples after this (defined as estimated ELISA sensitivity), depended on the cut-off value in the specific ELISAs. Cut-off values from pre- and early post-infection samples of individual animals demonstrated a seroconversion in all ELISAs on average after 10-15 days post-infection (dpi). The AB detection was delayed in the T.c. native and denatured AG-based ELISAs using cut-off points from uninfected cohort cattle (16.5 dpi, 19.3 dpi) and the adult cattle population (22.1 dpi, 25.0 dpi). The T.v. AG-based ELISAs however lacked crossreactiviy to T.c. ABs. The estimated sensitivity of each T.c. AG-based ELISA was above 96% throughout, but significantly lower for the T.c. native AG-based ELISA (91.1%) when the adult cattle derived cut-off point was used (p<0.01). The sensitivity of the phase contrast buffy coat technique was similar to the T.c. AG-based ELISAs, but significantly lower when the T.c. denatured AG-based ELISA was used at the adult cattle derived cut-off point (p<0.05). The implications of the results and future research aspects on ELISAs to detect trypanosomal ABs and AGs are discussed.

Identification of unacceptable background caused by non-specific protein adsorption to the plastic surface of 96-well immunoassay plates using a standardized enzyme-linked immunosorbent assay procedure.

A standardized enzyme-linked immunosorbent assay (ELISA) was used to examine the capacity of immunoassay plates to prevent non-specific protein binding under blocking conditions. Data from 16 types of 96-well microtitre plate from seven commercial sources, are described. Plates were evaluated with respect to their capacity to adsorb a conjugated antibody in diluent buffer containing non-ionic detergent Tween 20 (0.05%) and skimmed milk proteins (5%). Plates with an absorbance value of > or = 0.05, in not more than one well, were defined as within acceptable limits. Major problems were seen in high binding gamma-irradiated polystyrene plates, from all sources, where only < or = 30% of plates were acceptable. These showed high, randomly distributed, non-specific binding, with some wells showing absorbance values > 2.0. Similar results were obtained when high binding plates were repeatedly gamma-irradiated, and after gamma-irradiation of low binding polystyrene plates. For high binding, non- gamma-irradiated polystyrene plates, approximately 70% of plates were acceptable. Better results (86-100% acceptability) were observed for all low binding polystyrene plates. Only one source in three provided acceptable, low binding, polyvinylchloride plates. This paper confirms a widely held view that non-specific binding to certain plates could be a serious factor in both the development and application of ELISAs. Therefore, the test protocol described is proposed as an additional quality control method for certifying ELISA plates by commercial companies.

Pitfalls in the application of enzyme-linked immunoassays for the detection of circulating trypanosomal antigens in serum samples.

The experimental infection of two goats with Trypanosoma vivax trypanosomes provided samples for analysis using parasitology techniques and antigen-detection enzyme-linked immunosorbent assays (ELISAs) for T. vivax, T. congolense and T. brucei. Clinical, parasitological and serological findings were monitored during the course of infection to identify problems in the application of these ELISAs. The data clearly showed that the ELISAs examined were entirely unsuitable for the reliable detection of trypanosomal antigen. Consequently, research strategies pertinent to the development of a new generation of both antigen and antibody ELISAs are outlined considering the problems encountered. These were (1) the reactivity of the reagents; (2) the specificity of the reagents; (3) the nature of the test sample, e.g. the compartmentalisation of trypanosomes between plasma, serum and red blood cells; (4) possible interference with the ELISA through immune complexing; and (5) the biology of the host/trypanosome relationship to gain an understanding of fluctuations in trypanosomes in the systemic circulation.

Improved methods for the diagnosis of African trypanosomosis.

The diagnosis of trypanosomosis in animals with low parasitaemia is hampered by low diagnostic sensitivity of traditional detection methods. An immunodiagnostic method based on a direct sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies, has been examined in a number of African laboratories for its suitability for monitoring tsetse control and eradication programmes. Generally, the direct sandwich ELISAs for the detection of trypanosomal antigens in serum samples have proved to be unsatisfactory with respect to diagnostic sensitivity when compared with traditional parasitological methods such as the dark ground/phase contrast buffy-coat technique. Consequently, antigen-detection systems exploiting various other direct, indirect and sandwich ELISA systems and sets of reagents are being developed to improve diagnosis. In addition, an existing indirect ELISA for the detection of antibodies has been improved and is being evaluated in the field in order to detect cattle that are or have been recently infected with trypanosomes. Developments and advantages of other diagnostic techniques, such as dip-stick assay and tests based on the polymerase chain reaction are also considered.

Detection of viruses in livestock.

Diseases caused by viruses are a constant and major problem for livestock production world wide. The diseases range from highly contagious acute forms with high mortality, to chronic disabling diseases with an insidious effect on production. Such diseases cannot be regarded as static in nature due to the highly mutable nature of viruses and their direct selection at the host level and indirect selection in vectors, inducing changes in pathogenicity. Considerable efforts are needed to control these diseases including accurate and rapid diagnosis using both classical and emerging technologies. The methods used are based on both serological and molecular biological based methods. The ELISA and use of monoclonal antibodies are significant in the serological field whereas the Polymerase Chain reaction (PCR) and its direct and indirect uses for identifying (sequencing) and amplification of gene products, is vital to both research and applied fields. Both areas have to be used in a complementary way in the diagnosis of virus diseases.

Rinderpest: at war with the disease of war.

Rinderpest, the legendary cattle plague, has caused devastating losses for centuries and remains the biggest threat to sustainable livestock production in developing countries. Strenuous efforts are now being made to achieve global eradication of this viral disease, a goal made feasible through the use of attenuated live vaccines developed in the 1950s. Their use almost resulted in eradication in the 1970s but left several foci of disease from where the plague then re-emerged. Recent mass vaccination has resulted in the limitation of disease to parts of Africa, Pakistan, Afghanistan, the Middle East and India. Confirmation of the disease status of the countries has been aided by developments in serological techniques through exploitation of monoclonal antibodies (in Enzyme Linked Immunosorbent Assay-ELISA) and by advances in molecular biology such as in the use of polymerase reaction technologies (PCR). This has extended into the development of new recombinant vaccines. It is anticipated that eradication will be complete by the year 2010. This would be only the second example, after smallpox in man, of the eradication of a viral disease. The picture shows a scene of devastation during the Great Rinderpest Pandemic of 1889-1897 in South Africa. The disease swept through the African continent killing virtually all the cattle and wild ungulates.

Serological study of type A Indian foot-and-mouth disease virus isolates.

The antigenic relationship of sixty type A foot-and-mouth disease (FMD) viruses isolated between 1968 and 1993 has been determined with reference to a post-vaccinal bovine serum produced against type A IND 17/82. A micro-neutralization test and ELISA were used to compare isolates. Analysis of the results indicated that there was a positive correlation between the data from the two methods. The study indicated that type A IND 17/82 had a broad immunogenic spectrum and could be considered as a candidate vaccine strain for incorporation in FMD vaccines in India.

Antigenic analysis of SAT 2 serotype foot-and-mouth disease virus isolates from Zimbabwe using monoclonal antibodies.

This paper compares strains of foot-and-mouth disease (FMD) serotype SAT (South African Territories) 2 viruses isolated from Zimbabwe and other African countries using monoclonal antibodies (MAb). A sandwich-ELISA was used to examine the relative binding of anti-SAT 2 MAb to the various viruses. The MAb-binding profiles of viruses isolated from field samples were compared using hierarchical cluster analysis. Viruses were obtained from game animals, mainly African buffalo (Syncerus caffer) which is the natural host and reservoir for SAT serotypes in Africa, and from cattle showing clinical signs of FMD, as well as from animals suspected of carrying the virus subclinically. Some isolates have been adapted for use as vaccine strains. The results showed that most of the Zimbabwe isolates collected between 1989 and 1992 were an antigenically closely-related group. Although differences were observed between Zimbabwe isolates collected between 1989 and 1992 and those collected in 1987, there was no correlation with the different MAb binding patterns within the 1987 group and the epidemiological information received from the field. Similar profiles were observed for many SAT 2 viruses, including viruses isolated over a 50-year period and from geographically distant areas. This indicates an inherent stability in antigenic profiles of SAT 2 viruses. The MAb panel was capable of assessing antigenic variation, since very different profiles were obtained for some isolates. The work also allowed comparison and characterization of anti-type SAT 2 MAb from different laboratories. The findings are discussed with reference to selection of vaccine strains.

Quantification of whole virus particles (146S) of foot-and-mouth disease virus in the presence of virus subunits (12S), using monoclonal antibodies in a sandwich ELISA.

This paper describes a method for the specific quantification of whole virions of foot-and-mouth disease (146S) in the presence of virus subunits (12S). The method involves the use of virus neutralising monoclonal antibodies directed against a linear epitope of the VP1 loop region of a type O virus. The monoclonal antibodies were used as both capture and detecting reagents (labelled with horse radish peroxidase) in a sandwich ELISA. Such monoclonal antibodies also have the advantage that they do not detect viruses containing proteolytically cleaved VP1, thus the assay system is ideal for estimation of whole particles in vaccine manufacture where the immunogenicity of the vaccine depends on virus integrity (whole virions being present) and uncleaved capsid protein. VP1. Other combinations of different anti-type O FMD virus monoclonal antibodies used as capture and detecting reagents were also examined. The system could be adapted to on-line continuous testing of virus being produced during a manufacturing run allowing maximisation of virus yield and quality control.