John R. Crowther

The ELISA guidebook

1) Overview of ELISA in Relation to Other Disciplines 2) Systems in ELISA 3) Stages in ELISA 4) Titration of Reagents 5) Theoretical Considerations 6) Practical Exercises 7) Monoclonal Antibodies 8) Validation of Diagnostic Tests for Infectious Diseases 9) Charting Methods for Internal Quality Control for Competition ELISA 10) Charting Methods for Internal Quality Control of Indirect ELISA 11) Ruggedness and Robustness of Tests: Aspects of Kit Use and Validation 12) More Advanced Statistical Methods for Quality Assurance, Test Validation and Interpretation 13) Internal Quality Control and External Quality Management of Data in Practice 14) Immunochemical Techniques 15) Test Questions

A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus: II. Application

The liquid-phase blocking sandwich ELISA has been evaluated for the serological study of antibodies against foot-and-mouth disease virus (FMDV). The titres recorded for sera from a population of more than 300 British uninfected, unvaccinated cattle which were examined against each of the seven immunologically distinct FMDV types were less than 1 in 40. A positive correlation between ELISA and VN titres was recorded for sera either vaccinated or involved in outbreaks of FMDV. The overall regression between the ELISA/VN data showed that 1 in 16 by VN was equivalent to 1 in 40 by ELISA. Thus ELISA is sensitive, specific and reproducible and results may be directly correlated to those recorded by VN. Serum titres may be interpreted as positive or negative and the number of sera which require retesting would be considerably less than by VN. It is suggested that this ELISA may be used as an alternative to the existing VN test for the quantification of antibodies against FMD virus.

Alteration in antibody reactivity with foot-and-mouth disease virus (FMDV) 146S antigen before and after binding to a solid phase or complexing with specific antibody

This paper describes the reactions of a number of monoclonal antibodies produced against purified whole virions of foot-and-mouth disease virus in 3 different enzyme immunoassay systems. The first system used whole virus bound non-covalently to microplates; the second used whole virus trapped by a polyclonal antibody which was bound to microplates; and the third allowed the monoclonal antibodies to react with the whole virions in suspension (liquid phase) before trapping by the solid-phase-bound polyclonal antibody. Different reactions with panels of monoclonal antibodies were observed depending on which system was used. Such variations in reactivity give an insight into the alterations in the expression of virus epitopes in the different enzyme immunoassay systems. The reactions of selected monoclonal antibodies were used to illustrate these changes and the results compared to those obtained in similar systems using polyclonal antisera produced against isolated virion polypeptides.

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.

Early, rapid and sensitive veterinary molecular diagnostics - real time PCR applications

Preface.- acknowledgements.- chapter 1: background.- 1. aims of this book, 2. what is pcr?, 3. what is the use of pcr?. 4. pcr and infectious diseases -the veterinary picture, 5. laboratory diagnostic technology, 6. references.- chapter two: traditional pcr, 1. traditional pcr, 2. pcr reaction, 3. pcr set up and optimization, 4. the pcr plateau effect, 5. radioisotope-pcr based methods, 6. references.- chapter three: real time pcr. the basic principles: 1. traditional pcr versus real time pcr, 2. optimising a real time pcr reaction, 3. references.- chapter four: new trends in the diagnosis and molecular epidemiology of viral diseases: 1. background, 2. pcr methods used in routine molecular diagnostics, 3. acknowledgements, 4. references.- chapter five: disease diagnosis using real-time pcr specific procedures for important veterinary pathogens.- chapter six: pcr laboratory set-up: 1. establishment of a pcr laboratory.- 2. quality assurance programme or accreditation.- 3. references.- chapter seven: analysis and troublesho0ting.- chapter eight: specifications for pcr machines.- glossary of terms.- index.

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.

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

Abstract 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.

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.