ELISA assay as a diagnostic tool (2/2): Interpreting results

Considerations in interpreting results:

• It takes time for pigs exposed to a pathogen (wildtype or vaccine) to produce enough antibodies to be detected by ELISA (usually at least 2-4 weeks post-exposure depending on pathogen and targeted antibody)
• Antibody ELISA assays cannot differentiate maternal antibodies (passive antibodies) from antibodies due to exposure (active antibodies).
• Antibody ELISA assays often cannot differentiate antibodies due to vaccine from those due to wildtype exposure
• In few instances, special ELISA assays can be developed to differentiate infected from vaccinated animals (DIVA vaccines)
• Antibody ELISA results do not imply protection. Protection can come from different antibodies which may not be measured by the assay or by cell mediated immunity which is completely different than humoral or antibody mediated immunity and is not measured by the ELISA assay.
• Antigen ELISA is significantly less sensitive (low analytical sensitivity) in detecting antigen as compared to PCR. PCR assay actively replicates the DNA/RNA present and therefore can detect extremely low levels of viruses or bacteria. On the other hand, antigen ELISA require a significant greater concentration of the virus or bacteria to be able to create enough of a color change that it can be detected.
• Results are standardized and commonly consistent across laboratories when using commercially produced ELISA kits (commonly available and used). In-house laboratory ELISA assays can produce great variability in results unless the production practice is highly standardized and followed.
• Not all pathogens produce a measurable immune response
• Each ELISA kit will usually test for different proteins which can produce vastly different results for the same sample. A notable example for this is the many differences in Mycoplasma hyopnemoniae antibody ELISA kits available; definitely not all kits are created equally.
• Dose and/or route of exposure can impact the level of immune response (level of antibody production)
• Important to know the reported assay sensitivity (ability to detect true positives) and specificity (ability to identify true negatives as negatives; no cross reactions)
• False positive and false negative results can occur as amount of exposure and immune response to pathogens vary between individual pigs (normal distribution) and often there is some background or cross reactivity that cannot be completely eliminated (See Figure 1)
• Antibodies usually remain present/measurable for several months after exposure/vaccination

Negative result

• Sample/herd truly negative for pathogen (no exposure and no vaccine)
• Sample herd truly negative for pathogen although animals may be vaccinated (if assay has DIVA capabilities) (Antibody ELISA only)
• Not enough antigen present in the sample for detection (Antigen ELISA only)
• Sample collected too early in the disease and the pig has not been able to mount a detectable immune response (Antibody ELISA only)
  • PRRS often requires 7-10 days for seroconversion to occur
  • Lawsonia intracellularis often requires 4-6 weeks for seroconversion to occur depending on dose of exposure
• Sample collected too late in the disease and the pathogen is not present anymore.
  • Influenza virus present in nasal secretions only for first 3-4 days
• Targeted antibody/antigen mismatch
  • Different Influenza strains (Differences between H1N1 and H3N3 and also differences between different clads of H1N1)
• Low prevalence in the herd and animal(s) sampled were not infected
  • Need to increase the number of animals sampled
• Particular disease may not be stimulating enough antibodies for detection
  • Mycoplasma hyopneumoniae is mostly attached to cilia in lung and do not stimulate high production of IgG in serum
    • Recent field infection produce much stronger ELISA results
  • Mycoplasma hyopneumoniae vaccines do not stimulate a strong IgG immune response
    • Different ELISA assays detect vaccines differently
    • Usually only about 30% of vaccinated animals will seroconvert
• Dilution of a weak positive sample due to pooling
  • Strongly discourage pooling of samples!

Positive sample

• Sample/herd truly positive for pathogen exposure
• Unable to differentiate if sample is infectious or non-infectious
• Unable to differentiate maternal antibodies from natural exposure or vaccination
  • Most maternal antibodies disappear by 8-12 weeks of age
  • As pigs get older, ELISA values should significantly decrease over time
  • Can test pigs over time to confirm maternal antibody decay as well as lack of field exposure (no new infections)
• Depending on targeted pathogen, detection of protein (antigen/antibody) does not always confirm disease
  • Serum that tests antibody ELISA positive for porcine circovirus type 2 (PCV2), confirms exposure to PCV2 both to vaccinated or infected animals (both which are quite common) but does not confirm whether wasting or disease is attributable to PCV2 or whether vaccine failure has occurred. Need to do immunohistopathology of lung and or lymph tissue to demonstrate disease associated with PCV2.
  • Assay may not be able to differentiate exposure to vaccine virus/bacteria from modified live vaccine from wildtype infection (Antibody ELISA only)
    • Applies to both killed and modified live vaccines
    • Helpful to know the timing of vaccine used although not critical as animals can remain positive for some diseases for a long period of time (many months)
• Cross contamination is unlikely as it takes a large amount of contamination to produce a positive result (concentration dependent)

• A single ELISA value may not fully identify the stage of infection (need clinical history)
  • See Figure 2
  • X = ELISA value
  • Three different points in an outbreak can produce the same ELISA value yet the interpretation and actions needed would be quite different:
    • Point A – Ealy in infection – outbreak is starting; actionable
    • Point B – Late in infection – outbreak is ending; not much to do at this point
    • Point C – Second outbreak – second outbreak is starting, biosecurity practices/ reason for re-break must be addressed
  • A second sample collection from same animals 10 days to 2 weeks later may be needed to further determine stage of outbreak (along with clinical history)
    • If second sample results are higher, then likely at point A
    • If second sample results are dramatically higher, then likely at point C
    • If second sample results do not change the likely in a plateau phase
    • If second sample results go down, then likely at point B
• High S:P values can often be associated with more recent exposure
  • Especially after a second exposure or booster vaccination (See Figure 2) (Antibody ELISA only)
  • It is important to note that just because an infection is recent it does not imply the pathogen is still present (i.e., animal is viremic or bacteremic) (Antibody ELISA only)
    • This is especially true for Porcine Reproductive and Respiratory Syndrome (PRRS) virus.
      • Pigs can be ELISA negative and yet viremic
      • Pigs can have high antibody ELISA values and not be viremic
      • PCR assay is used to establish viremia status of animals
• Low S:P values
  • Particular disease may not be stimulating enough antibodies for detection
    • Mycoplasma hyopneumoniae is mostly attached to cilia in lung and do not stimulate high production of IgG in serum
      • Recent field infection produces much stronger ELISA results
    • Mycoplasma hyopneumoniae vaccines do not stimulate a strong IgG immune response
      • Different ELISA assays detect vaccines differently
      • Usually only about 30% of vaccinated animals will seroconvert
  • Pigs being treated with antibiotics for several weeks may suppress bacterial growth during that time, thus decreasing disease exposure (but not eliminating) resulting in a slower or weaker immune response to exposure
    • Using antibiotics against Mycoplasma hyopneumoniae in early and mid-nursery stages
    • Using antibiotics against enteric pathogens such as Lawsonia intracellularis in early and mid- nursey stages