How does a lab test measure Rheumatoid Factor (RF) Immunoglobulin A (IgA), Immunoglobulin M (IgM), and Immunoglobulin G (IgG)?

Laboratory Testing for Rheumatoid Factor (RF) IgA, IgM, and IgG

Rheumatoid factor testing for IgA, IgM, and IgG isotypes is performed using enzyme-linked immunosorbent assay (ELISA) methodology, with rabbit IgG as the solid-phase antigen and isotype-specific detection antibodies, requiring pepsin digestion of serum to accurately measure IgG RF without interference from IgM RF. 1, 2, 3

Standard ELISA Methodology

Antigen Preparation and Detection System

• The assay uses rabbit IgG (or a complex of human serum albumin with rabbit IgG anti-HSA antibodies) as the target antigen attached to the solid phase 2, 3
• Isotype-specific detection is achieved using enzyme-conjugated antibodies: anti-human IgM (Fc5μ specific), anti-human IgG [F(ab')2], and anti-human IgA (alpha chain specific) 3
• Some laboratories employ biotinylated monoclonal antibodies specific for each isotype, followed by alkaline phosphatase-conjugated streptavidin and substrate for signal detection 2

Critical Pre-Treatment for IgG RF Measurement

• Pepsin digestion of serum is mandatory before measuring IgG RF to eliminate false-positive results caused by IgM RF interference 1, 2, 3
• Without pepsin treatment, high-affinity IgM RF can displace IgG RF from the antigen, causing false-negative results, or IgM anti-IgG Fc rheumatoid factors can bind and create false-positive results 4, 1
• After pepsin digestion, IgM RF is destroyed while IgG RF remains intact and can be specifically measured without interference from IgA RF 3

Alternative Testing Methods

Nephelometry

• Rate nephelometry provides rapid, quantitative results reported in international units/mL and is considered the easiest and quickest method to perform 1, 5
• Nephelometry shows good correlation with ELISA for IgM RF measurement, though ELISA demonstrates greater sensitivity 5
• This method is primarily used for IgM RF quantitation and does not routinely measure IgG or IgA isotypes 1

Traditional Agglutination Methods

• The Rose-Waaler test (sheep red blood cell agglutination) is the least sensitive assay and the most difficult to perform and interpret, now largely obsolete 1
• Latex agglutination tests are sensitive and widely used for screening, showing good correlation with ELISA (r = 0.82), though ELISA detects additional positive cases 2, 5

Clinical Performance Characteristics

Sensitivity and Specificity by Isotype Combination

• Testing IgM RF alone provides 91% sensitivity, 76% specificity, and 62% predictive value for rheumatoid arthritis 3
• Combining IgM + IgA RF testing improves performance to 79% sensitivity, 89% specificity, and 80% predictive value 3
• Testing all three isotypes (IgM + IgG + IgA) achieves 53% sensitivity but 99% specificity and 96% predictive value for RA 3
• When patients have all three isotypes plus >150 U of IgM and/or IgA, the clinical variables reach 70% sensitivity, 97% specificity, and 93% predictive value 3

Isotype-Specific Findings

• IgG RF is found only in association with IgM RF—there is no "hidden" IgG RF occurring independently 3
• IgA RF is always accompanied by IgM RF in rheumatoid arthritis patients 3
• Many latex-RF negative RA patients have elevated ELISA levels of IgM RF, suggesting the assay reveals non-agglutinating or "hidden" IgM RF 6
• IgA RF levels correlate with IgA-containing immune complexes and are elevated in both latex-RF positive and negative RA patients 6

Important Technical Considerations

Avoiding False Results

• Fc-specific (or IgG1-specific) secondary antibodies are highly recommended to avoid cross-reactivity with co-binding IgM and IgA antibodies 4
• The screening test for IgG RF requires confirmation by pepsin digestion to discriminate between false and true IgG-RF—in one study, 42 sera screened positive but only 25 (39%) were confirmed as true IgG-RF positive after pepsin treatment 2
• IgM antibodies (whether autoantibodies or anti-HLA) can cause positive results but are not clinically relevant for transplantation contexts 4

Quality Control and Standardization

• Both ELISA and nephelometry are reliable, accurate, rapid, and sensitive for routine clinical purposes 5
• ELISA is applicable to routine testing of large numbers of sera and provides quantitative results 2
• Good correlation exists between all assay methods (ELISA, nephelometry, latex agglutination, Rose-Waaler), though sensitivity varies 1, 5

Clinical Context and Interpretation

Disease-Specific Patterns

• In rheumatoid arthritis, all three RF isotypes show continuous decline during treatment with gold salts, making serial testing useful for monitoring 3
• IgA RF are elevated in many RA patients regardless of latex-RF status, and their increase is not merely an expression of polyclonal B-cell activation 6
• In systemic lupus erythematosus, only some cases (mainly latex-RF positive) show enhanced IgM RF on ELISA, and IgA RF values remain normal even in latex-RF positive patients 6

Limitations of IgG RF Measurement

• Quantitatively, IgG RF does not play a significant diagnostic role for RA because values are widely dispersed in normal subjects and the mean increase in RA patients is relatively small 6
• Some overlap between patient and control sera occurs with all assays, including ELISA 1