Have you previously encountered false-positive results in your ELISA? Let us present Mabtech's answer for cytokine quantification in plasma and serum containing rheumatoid factor (RF).
How rheumatoid factor makes ELISA results inaccurate
In a sandwich ELISA, matched antibody pairs are used for the capture and detection of soluble analytes. When the analyte is present in the sample, a positive signal is measured (Fig. 1A). However, interfering factors in serum and plasma, such as rheumatoid factor (RF) and heterophilic antibodies, can crosslink the assay antibodies, which causes false-positive signals and inaccurate analyte quantification (Gehin et al. 2021) (Fig 1B). Cross-linking makes it impossible to distinguish between specific and non-specific signals and, ultimately, renders the results unreliable. Extra sample pre-treatment steps, for example, antibody removal by PEG 6000 precipitation or immunoglobulin blockade, have previously been suggested to reduce this interference (Bartels et al. 2011, Churchman et al. 2012).
Eliminate interference from heterophilic antibodies by choosing ELISA PathRF
RF was first found in patients with rheumatoid arthritis (RA) and has since been described in many conditions, including cancer, chronic infections, sarcoidosis, mixed connective tissue disease, Sjögren’s syndrome, and systemic lupus erythematosus (Ingegnoli et al. 2013, Tiwari et al. 2021). Therefore, the risk for false-positive results caused by the interfering RF and heterophilic antibodies is higher in samples from patients than in samples from “healthy controls” (Kharlamova et al. 2021).
To ensure that your data can be compared across different cohorts, consider switching to ELISA PathRF (Fig. 1C). In this ELISA format, the original antibody pair that specifically detects the analyte is still present but by introducing a recombinant backbone to the detection antibody and adding a specialized RF-block diluent, the risk of interference is circumvented.
A) Principle of a sandwich ELISA.
B) Interference by heterophilic antibodies and RF causing a false-positive signal.
C) Circumventing interference with an ELISA PathRF kit.
What is RF?
RF (rheumatoid factor) is a variable mixture of many different antibodies; some of these antibodies bind other antibodies and some are autoantibodies (antibodies that bind self-antigens).
What are heterophilic antibodies?
Heterophilic antibodies bind other antibodies. In human samples, these are either autoantibodies that bind human immunoglobulins or human anti-animal immunoglobulin antibodies (HAIA), including human anti-mouse antibodies (HAMA).
Our ELISA PathRF kits are validated with RF‑containing plasma
An unmatched set of antibodies can be used to determine whether a signal obtained in an ELISA is a true or a false-positive signal. Such an unmatched set consists of capture and detection antibodies that bind to entirely different proteins. This unmatched set cannot result in a true signal. If, however, a signal is obtained, this shows that factors in the sample cross-link the capture and detection antibodies creating a false-positive signal.
In 100 plasma samples from individuals with rheumatoid arthritis, we observed a high frequency of false-positive results using an unmatched ELISA (Fig. 2). By contrast, the ELISA PathRF resulted in no false-positive signals (Fig. 2).
Figure 2. Plasma from patients with RA (n=100) was measured in an unmatched ELISA (unmatched set of antibodies against different targets) or with an unmatched ELISA PathRF.
- Bartels et al., Rheumatoid factor and its interference with cytokine measurements: problems and solutions, Arthritis 2011
- Churchman et al., Multiplexing immunoassays for cytokine detection in the serum of patients with rheumatoid arthritis: lack of sensitivity and interference by rheumatoid factor, Clin Exp Rheumatol 2012
- Gehin et al., Rheumatoid factor and falsely elevated results in commercial immunoassays: data from an early arthritis cohort, Rheumatol Int 2021
- Ingegnoli et al., Rheumatoid Factors: Clinical Applications, Dis Markers 2013
- Kharlamova et al., False Positive Results in SARS-CoV-2 Serological Tests for Samples From Patients With Chronic Inflammatory Diseases, Front Immunol 2021
- Tiwari et al., Rheumatoid Factor, Treasure Island (FL): StatPearls Publishing; 2021 Jan
Curious to dive even deeper into heterophilic antibodies and rheumatoid factor? Watch our webinar!