Methodologies

• Two tests are recommended for detection of serum or synovial fluid rheumatoid factor in veterinary species.
• The most commonly used test is theRose Waller Assay:
  • In this test, a washed suspension of sheep or canine erythrocytes are preincubated with an antiserum specific for sheep or dog erythrocytes.
  • The antiserum must be used at a sub-agglutinating dose, so that the red cells are coated with antibody but do not agglutinate.
  • This preparation of antibody-coated red blood cells (RBC) is then incubated with serum or synovial fluid from the patient.
  • The test samples are serially diluted, usually in a microtiter system and a standard volume of antibody-coated RBC suspension is added to each dilution.
  • The antibody-RBC complex mimics the antibody-antigen complex to which RF binds in vivo.
  • If RF is present in the serum or synovial fluid sample it will bind to the anti-RBC antibody and cause cross-linking of these molecules, and thus agglutination of the red cells.
  • This agglutination is scored visually, and the last well in the microtiter plate in which agglutination has occurred is recorded in order to calculate the titer of the reaction.
  • A duplicate titration of the sample is incubated with a suspension of RBC that has not been pre-incubated with anti-RBC antibody as a negative control.
• The second method used for detection of RF isELISA  Enzyme linked immunosorbent assay (ELISA)  :
  • In this assay, a purified immunoglobulin is coated to the plastic wells of the ELISA microplate.
  • Serum or synovial fluid from the patient is titrated into replicate wells, and if RF is present it will bind to the immunoglobulin.
  • Presumptively, the process of immunoglobulin adhering to the plastic surface of the ELISA plate is sufficient to alter the conformation of the molecule to permit RF binding.
  • Any bound RF is subsequently detected using an antiserum, eg specific for canine IgM, that is conjugated to an enzyme (typically alkaline phosphatase   Blood: biochemistry - alkaline phosphatase (SAP, ALP)  ).
  • The appropriate substrate is added and the ensuing color change is recorded spectrophotometrically.

Control

• In addition to the internal control for RF binding (non-coated RBC), the RF assay will include a negative and positive control serum of known RF content.
• The positive control serum will produce an agglutination reaction with antibody-coated RBC, but not uncoated RBC.
• In the ELISA method, the positive control serum may be used to generate a standard curve, the slope of which can be compared to those obtained with test sera for calculation of the result.

Availability

• The RF assay will usually only be available from specialist diagnostic or research laboratories.

Validity

Sensitivity

• The ELISA is a more sensitive test than the Rose Waller Assay, however the ELISA is generally used as a research tool rather than a routine diagnostic test.

Specificity

• RF is a non-specific autoantibody.
• It may be present in clinically normal animals, or in animals with non-autoimmune disease.
• In most cases these will be relatively low-titered RFs.