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CHAPTER 1 - TECHNICAL BACKGROUND TO THE INDUSTRY
This chapter introduces and compares two in vitro testing technologies – immunoassays and nucleic acid testing (NAT) – and their commercial applications in the animal health and breeding industries. The chapter commences with a review of conventional diagnostics traditional immunoassay designs. This is followed by a discussion of more recently introduced rapid immunoassays suitable for animal-site use. The principles of probe-based NAT protocols are outlined. Traditional and emerging NAT formats and approaches to infectious disease testing, genotyping, and identity testing are also reviewed.
1.1 Immunoassays
1.1.1 Introduction
Immunoassays are chemical analyses using diseases and antibodies. They use disease-causing agents (called ‘antigens’) and their corresponding antibodies to detect and analyse various chemical substances in animals, food, etc. The intention is to identify whether a substance, and thus a disease, is present. There are two general approaches to diagnosing diseases by immunoassays:
• testing for specific antigens using monoclonal antibodies
• testing for antigen-specific antibodies using recombinant antigens or synthetic peptides
Measuring techniques based on the detection of antigen-specific antibodies are often collectively termed serology.
Immunoassays may be classified by the type of label used, eg:
• radioisotopes (radioimmunoassays, RIAs)
• enzymes (enzyme immunoassays, EIAs)
• fluorescent labels (fluoroimmunoassays, FIAs)
• luminescent labels (chemiluminescent immunoassays, CLIAs)
In EIAs, the final step is the chromogenic reaction of the enzyme with its substrate.
When testing for antigens, the monoclonal antibodies are usually covalently attached (coated) onto a solid phase support (ie a physical substance) such as polystyrene microplate wells (tubes, beads, or magnetic particles may also be used). EIAs making use of solid phase support are called enzyme-linked immunosorbent assays (ELISAs).
In ELISA-based serological testing, the solid phase is coated with antigens. Virus infection can often be detected by demonstrating a rise in the serum antibody titre to the virus between acute- and convalescent-phase serum samples (seroconversion). Paired serum samples can be evaluated for their virus antigen-specific antibody titres by ELISA.
ELISA assays have become one of the most popular methods for quantification of analytes in samples because, in addition to their sensitivity and specificity, they are simpler and less costly than most other analyses.
Immunohistochemistry is a valuable diagnostic tool used in histopathology and cytopathology. The technique involves the use of specific antibodies to reveal the presence of particular antigens in tissue or cells. Positive immunoreactivity is normally detected by using a second layer of antibodies labelled with fluorescein (indirect immunofluorescence).
1.1.2 Competitive immunoassays
The most basic assay design is the competitive immunoassay. Competitive ELISAs are frequently used for the detection of small analyte antigens containing a single epitope.
In a competitive ELISA a carefully titrated concentration of antibody is coated onto the inside wall of the microwell. In a single reaction, antigen from the test sample and the enzyme-labelled antigen conjugate compete for a limited number of immobilised antibody-binding sites. The amount of antibody-antigen-enzyme complex bound to the solid phase (microwell) is inversely related to the concentration of antigen present in the sample.
1.1.3 Sandwich immunoassays
Competitive immunoassays have a number of drawbacks, most notably a restricted working range and limited sensitivity. These problems have been substantially overcome, for large analytes at least (such as serum proteins and hormones), by the development of two-site immunometric or ‘sandwich’ assays. In these assays there are two antibodies which bind the analyte independently, ie at different epitope sites. Immunometric assays may, depending on the choice of label, be described as immunoradiometric (IRMA), immunoenzymometric (IEMA), immuofluorometric (IFMA), or immuochemiluminometric (ILMA). The most widely used format are sandwich ELISAs.
In a sandwich ELISA, the sample is incubated in a microwell precoated with capture antibody. Antigen in the sample bind with the capture antibody and becomes immobilised. The microwell is washed to remove unbound antigen and enzyme conjugate is added. The antibody of the enzyme conjugate binds with the immobilised antigen to form a sandwich of antibody-antigen-antibody/enzyme bound to the microwell. The amount of enzyme bound to the microwell is directly proportional to the amount of antigen in the sample. ELISA sandwich methodology can also be used for the detection of serum antibodies specific to certain infectious diseases.
Sandwich assays are readily configured as single-use rapid immunochromato¬graphic devices.
1.1.4 Animal-site rapid immunoassays
Rapid immunoassays suitable for animal-site use (either in the clinic or in the field) are mainly membrane-based lateral flow immunochromatographic assays, although rapid membrane-based ELISA assays have also been developed (for example Idexx Laboratories’ SNAP PF heartworm antigen test kit).
In general, membrane format tests are less sensitive than microwell format tests, and tests using lateral flow immunochromatographic technology are less sensitive than tests using ELISA technology.
Due to their simplicity, utility and relatively low production costs, membrane-based lateral flow assays have, over the last decade, become an important tool not only in medical diagnostics but also in veterinary diagnostics and in the environmental and agricultural fields.
In chromatographic lateral flow devices, the sample is usually diluted with a special buffer and applied at a designated site, or the sample is applied first, followed by the buffer. The buffer contains all the reagents required for a competitive or an immunometric assay, including substrate (if it is an enzyme immunoassay). Traditionally colloidal gold conjugates were used, stored in a dry mobile state in the device. On coming into contact with the biological sample and buffer, the colloidal gold conjugate quickly becomes soluble and binds to antigen or antibody in the sample. It then moves across the membrane through capillary migration. If the colloidal gold has captured the specific antigen or antibody then a second antibody or antigen, immobilised at the test zone captures the colloidal gold-coupled immune complex. A pink/purple line appears in the test zone. The intensity of the colour developed may vary with the concentration of the antigen or antibody.
Capillary progression along the membrane can provide an initial sample purification step, and it may therefore be possible to use whole blood or milk samples.
Currently, most assay developers choose between gold particles (from 20nm to 45nm) and dyed polystyrene latex microspheres (from 150nm to 400nm). Dyed microspheres offer several advantages over the gold particles such as good lot to lot reproducibility, batch size of 1kg and more, uniform size distribution, ease of use for passive adsorption or covalent coupling, and above all, a range of more than 100 different colours. The last advantage is significant, and means that when several tests are run together the different analytes can be perfectly and easily identified.
Until recently, lateral flow assays have been used mainly for the qualitative detection of analytes. Increasingly, visual techniques are being replaced by fast and automated optical detection systems. Analysers have been designed to read strip-tests, based on colloidal gold or polystyrene microspheres labelled with different analytes, by remission or retransmission photometry.
1.2 Nucleic acid testing
1.2.1 Introduction
Nucleic acid testing (NAT) tests for specific DNA. They use single-stranded DNA probes designed to be complementary to the unique base sequence of the target nucleic acid. (NAT is sometimes also called DNA testing – DNA tests are the main type of nucleic-acid-based diagnostics). This matching DNA sequence may indicate (for example) the presence of an undesirable virus or bacterium, the presence of an allele coinherited with that for a genetic disorder, or the deleterious mutation itself. NAT provides a direct means of detecting nucleic acid sequences and thus allows detection of diseases at the genetic level.
DNA can be extracted from any nucleated cell. DNA is very stable and only the smallest quantities are needed for analysis. Sources such as blood, a hair follicle, and semen are commonly used.
1.2.2 Probe-based NAT protocols
Traditional probe-based NAT involves the following steps:
1. Sample preparation and nucleic acid purification.
2. Amplification of the target DNA sequence.
3. Labelling of the probe with a marker that generates a detectable signal upon hybridisation (ie the binding of the probe to the target DNA sequence).
4. Addition of the probe to the sample containing the DNA, and binding of the probe to the target DNA sequence, if present, to generate a detectable signal.
5. Amplification of the signal may also be performed. Signal amplification methods chemically enhance the detectable signal and offer an alternative to target amplification for increasing the sensitivity of NAT.
Currently, the most widely used method of DNA analysis is first, amplification of the target DNA and second, detection of the DNA with fluorescent dyes and fluorimeters. Roche’s polymerase chain reaction (PCR) is widely regarded as the gold standard amplification technique. PCR acts on a target molecule to generate a million or more copies of the target nucleic acid sequence through repeated cycles of heating and cooling.
In early PCR amplification processes, the nucleic acid in the sample was amplified first and then detected in a separate step. In 2001, Roche was awarded a US patent on a real-time PCR method that combines the two steps by including fluorescent dyes in the reaction, so that fluorescence increases as the number of nucleic acid copies increases throughout the reaction. Real-time PCR has made nucleic acid analysis faster and easier.
PCR tests are generally robust and accurate as long as appropriate techniques and controls are used. However, PCR is not widely available to companies developing commercial diagnostic tests due to licence restrictions. Several alternative target amplification technologies have been developed, many of which are isothermal and do not require expensive thermal cyclers, though they are currently being explored mainly for human diagnostic applications.
1.2.3 NAT formats
There are various processes, or formats, for performing probe-based tests. In certain formats, the probe is introduced to a target sample affixed to a solid matrix; in others, the probe is combined with the sample in solution (homogeneous assay). Solid matrix assays include:
1. In situ assays in which the probe reaction takes place directly on a microscope slide.
2. Dot blot assays in which the target DNA is fixed to a membrane. The DNA dot blot hybridisation technique is currently one of the most widely used systems assay systems.
3. Microplate and microarray assays in which the DNA is fixed on a solid surface and the reaction can be quantified by instrumentation. Hybridisation microarrays are flat chips that have different DNA probes located in known positions on the chip surface, usually a glass, plastic or silicon slide, in a grid-like formation. DNA microarrays offer the latest technological advancement for multi-gene detection and diagnostics.
1.2.4 NAT options
NAT techniques have brought tremendous changes in infectious disease testing. These changes are reflected in the sensitivity and specificity with which assays are able to detect pathogens in biological samples from animals during the course of the infection-disease process. Tests are typically probe-based, and use amplification to increase their sensitivity. Specific nucleic acid probes react with the genomic material of the pathogen in question. It is then amplified to produce DNA specific to the particular organism. To visualise, the amplified products are subjected to electrophoresis to measure size and migration pattern. Results can be made available in 24 hours or less. Furthermore, NAT can be used to detect viral latency or carriers (herpesvirus, FIV); viral shedding (coronavirus); to detect fastidious pathogens (Bartonella, Ehrlichia) in blood or body fluids; and to study the pathogenesis or to discover the etiologic agents of newly recognised infectious diseases.
Genotyping is the process of analysing locations within a genome where variations in a gene sequence, or genetic polymorphisms, are known to exist. Genotyping is performed using tests that rely on either DNA probe (hybridisation microarrays) or DNA sequencing technologies. DNA sequencing is labour-intensive, but is generally considered the most thorough and accurate method of genotyping. Genotyping tests are generally offered by specialist laboratories on a service basis. Genotyping can identify animals with specific genetic traits or disease predisposition. Carriers can be identified so that breeding programmes can be modified to control transmission of traits between generations.
The application of the DNA microsatellite technology has enabled accurate animal parentage analysis and individual identification. Microsatellites are short sequence motifs found in DNA, composed of repeating units of up to four nucleotides. By selecting several microsatellites, it is possible to generate a DNA fingerprint that is characteristic for a given animal. The DNA fingerprint produces a unique profile for each animal that not only determines parentage, but also can serve as a permanent identification record of the individual.
1.3 Immunoassays versus NAT
In infectious disease testing, antibody tests rely on antibody sensitivity and specificity, and on the presence of significant amounts of either the organism or the antibody. These factors result in a wide variability of results between samples. Nucleic acid-based tests offer greater specificity and sensitivity than immunoassays. The greater specificity of NAT compared with immunoassays arises from the fact that the diagnostic DNA probes and the target sequences that they recognise are usually dozens or hundreds of bases long whereas the region of an antibody that recognises its antigen consists of only a few amino acids. DNA methods have greater sensitivity because amplification techniques amplify target sequences or the analytical signal to such an extent that very small numbers of molecules – perhaps even a single organism – can be detected. It should also be noted that oligoncleotide probes are much easier to produce than monoclonal antibodies used in immunoassays.
Utilisation of nucleic acid testing has enabled infections to be detected during the preclinical stages. Through the detection of a particular infectious agent in animals with no clinical symptoms, the veterinarian now has the information necessary to take action to stop the spread of infection.
NAT can allow not only early detection of infection, but rapid speciation of organisms as well as strain typing for epidemiological analysis. Nearly any sample that could contain pathogens (virus, bacteria or parasites) can be analysed. Blood, urine, faeces, exudates, nasal secretions, intact tissues, and even environmental swabs have been used. Testing can be performed on samples obtained from live animals and necropsy specimens.
NAT are not affected by the presence of antibodies in the serum and can give an immediate indication of the effectiveness of a treatment protocol in clearing an organism. With immunoassays, vaccinated or previously exposed animals may give false positive results because of existing antibodies.
However, NAT is not useful in detecting antibody responses or seroconversion, or in managing endocrine disorders. Although the serological tests widely used in diagnostic virology have a major drawback due to the absence of antibodies in the early stages of infection (and occasionally also in the late stages of advanced disease), the presence of antibodies in the absence of any detectable infectious agent is a reliable indicator of past infection. It is often desirable to determine previous exposure to a virus or to assess the level of immunity following vaccination. Antigen tests only provide information on current infection (if a relevant antigen circulates in the blood of infected individuals, which does not always happen).
ELISAs can be designed to distinguish antibodies/titres due to natural infections from those due to vaccination. For example, in recent years, France, Belgium and the state of Iowa, US, concluded that vaccination would not suffice to eradicate pseudorabies completely and initiated elimination campaigns. Such elimination campaigns require a diagnostic test to differentiate between vaccinated and wild-type virus-exposed animals.
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