Antibodies - ELISA - PRRS diagnostics

PRRSv antibody detection is a constantly evolving diagnostic discipline.

Some techniques are associated with time and resource issues, and are now used primarily in research. These include immunoperoxidase monolayer assay (IPMA) a highly specific and sensitive method, and the first serological test reported for the diagnosis of PRRS. In this assay anti-PRRSv antibodies captured onto microtitre plates by virus-infected cells are detected by peroxidase-labelled secondary antibodies. Positive samples are then assessed by light microscopy. Similarly, virus neutralisation (VN) produces difficult to interpret results.
 
While antibody detection can identify new PRRSv infections, it is less suitable for monitoring previously infected herds since it is unable to distinguish between antibodies generated though new infection, previous infection or vaccination.
 
ELISA
The enzyme-linked immunosorbent assay (ELISA) is the most common assay for detection of PRRSv antibodies, and has a specificity as high as 99.9%, and a sensitivity of up to 98.8% (Iowa State University, 2010). The abundant N protein of PRRSv is used to capture anti-PRRSv antibodies from tissue samples onto the surface of coated test plates, and a peroxidase-conjugated secondary antibody is used to visualise captured anti-PRRSv. Several ELISA kits have been developed for specific purposes, including those to pick up early infection (detection of the early immunoglobulin IgM) and PRRSv isolates with nsp2 deletions.
 


While ELISA is both rapid and convenient, it has a number of limitations. Immune responses are typically variable between animals, and levels of antibody detected do not necessarily reflect the virulence of isolates. Furthermore, maternally derived antibodies and antibodies from feed can contaminate results, leading to false positive readings. False negative readings during early infections can also be an issue. For pigs that persistently become infected, viral detection techniques may be required.

FMIA
The fluorescent microsphere immunoassay (FMIA) is a multiplexed version of ELISA which uses differentially-dyed fluorescent beads to capture multiple anti-PRRSv antibodies, which are then labelled with fluorescently-labelled secondary antibodies. The technique allows several different antibodies to be detected simultaneously within a single, small sample. In serum, FMIA has demonstrated 98% sensitivity and 95% specificity. Furthermore, using oral fluids, for which traditional ELISAs have notoriously low sensitivity, FMIA has over 92% sensitivity and 95% specificity. It is also more sensitive than ELISA for detection of early infections.
 
Indirect IFA
The indirect fluorescent antibody (IFA) assay is similar to IPMA but uses fluorescently-labelled secondary antibodies to detect captured anti-PRRSv antibodies. Indirect IFA has demonstrated good specificity, but sensitivity depends on many factors, including the cell types used to capture antibodies, laboratory protocols, and the antigenic profile of anti-PRRSv antibodies. Typically, indirect IFA is performed after ELISA to confirm false-positive results, but has also been used in surveillance programmes using oral fluids and muscle transudates.
 
Field test strips
As is the case for viral detection, field test strips are now available to detect PRRSv-specific antibodies (using viral N protein). Field test strips are cheap, quick and easy to use without the need for expensive equipment, and have a reported sensitivity and specificity of 93% and 98%, respectively (Lyoo et al, 2005; Cui et al, 2008). However, the strips are not currently widely used, and as with other antibody detection assays, the kits may not be able to recognise antibodies of genetically diverse PRRSv.
 

Achieving a more complete diagnosis: isolate characterisation
It is often important to characterise the infecting virus isolate to inform management decisions such as whether to move animals onto another farm, to track isolate spread, and to determine if a new isolate might be present in a population of pigs. Molecular techniques, including RFLP and sequence analysis, have been used to provide a more definitive characterisation of the specific PRRSv detected by conventional assays.