A quantitative enzyme immunoassay for IgM rheumatoid factor using human immunoglobulin G as substrate

Area of Science:

• Immunology
• Biochemistry
• Clinical Chemistry

Background:

• Rheumatoid factor (RF) is a key biomarker in diagnosing autoimmune diseases like rheumatoid arthritis.
• Existing methods for RF detection, such as latex and bentonite flocculation, can lack sensitivity and standardization.
• A need exists for a more precise and reproducible quantitative assay for RF activity.

Purpose of the Study:

• To describe a novel quantitative "sandwich" enzyme-immunoassay (ELISA) for IgM rheumatoid factor (RF).
• To evaluate the sensitivity, reproducibility, specificity, and standardization capabilities of the developed ELISA method.
• To compare the performance of the ELISA using human versus goat immunoglobulin G (IgG) as substrate.

Main Methods:

• Development of a "sandwich" enzyme-immunoassay utilizing human immunoglobulin G (IgG) covalently bound to plastic discs as substrate.
• Quantification of RF activity using human IgG as substrate.
• Standardization of results using World Health Organization (WHO) reference RF serum, reported in IU/ml.
• Validation by comparison with latex and bentonite flocculation titrations.
• Confirmation of positive results using inhibition assays with aggregated and non-aggregated human IgG.

Main Results:

• The developed ELISA is a simple, sensitive, reproducible, and specific method for measuring RF activity.
• Results are standardized in IU/ml based on WHO reference serum, enabling consistent interlaboratory reporting.
• Interassay and interlaboratory variability were less than 7%.
• ELISA results showed good correlation with latex and bentonite flocculation titrations.
• The assay identified RF-positive specimens missed by latex and/or Waaler-Rose tests, confirmed by inhibition assays.
• Human IgG generally showed higher reactivity with RFs compared to goat IgG.

Conclusions:

• The described quantitative "sandwich" ELISA is a highly effective method for measuring IgM rheumatoid factor (RF) activity.
• The assay offers significant advantages in sensitivity, reproducibility, and standardization over traditional methods.
• The use of human IgG as substrate enhances reactivity, making it a preferred choice for RF detection.