Meridian is a primary manufacturer of specialized high-quality molecular reagents and offers solutions to a wide range of industries to diagnose and treat diseases, discover new therapeutics or develop tests for environmental, food and cosmetic safety.
However, endogenous antibodies present in some patient populations—particularly heterophilic antibodies and autoantibodies—can bind nonspecifically to these assay components, leading to erroneous results. While historically infrequent, such interference can have a disproportionate impact on diagnostic accuracy, clinical decision-making, and assay credibility. Notably, since the COVID-19 pandemic, reports of immunoassay interference have increased—likely due to elevated levels of circulating autoantibodies such as IgM rheumatoid factor. Reducing False Results in Immunoassay Testing Diagnostic immunoassays often rely on animal- derived antibodies to selectively detect disease- specific biomarkers in human samples.
The most well-known antibody interference is HAMA (human anti- mouse antibodies), which is due to the wide use of mouse monoclonal antibodies in diagnostic applications. However, HAMA only represents one type of heterophilic antibody (HA) interference - others include heterophilic antibodies to other animals such as goat (HAGA), sheep (HASA), and rabbit (HARA) which can cause false results when antibodies originating from these animals are used in immunoassays. In addition to HA there is another class of interference
called rheumatoid factor (RF), which is an autoantibody that reacts with the patient’s own immunoglobulin (Ig) and can cross-react with animal Ig, similar to HA/HAMA interference. To reduce interference from HA, HAMA and RF, IVD manufacturers commonly incorporate animal serum or purified animal IgG. These blockers work by binding to cross-reactive antibodies that might otherwise interact with assay antibodies derived from the same species. One example is mouse serum, or Mouse IgG, which is used to block
HAMA interference. Although Mouse IgG can be effective in blocking HAMA, it also has several limitations including: • Narrow coverage, only blocks HAMA and mouse-specific RF • Requires high concentrations to sufficiently block HAMA interference • Passive blocking mechanism - high concentration overwhelms the interfering antibodies and reduces the probability that they will bind to the detector or capture antibodies Mouse IgG and other immunoglobulins passively block HA interference by competitively binding to these interfering molecules with an affinity similar to that of the assay antibodies. This approach is particularly useful for mitigating human anti-mouse antibody (HAMA) effects. However, effective blocking often requires using these reagents at concentrations up to ten times higher than the assay antibodies, which can reduce signal strength and complicate miniaturized assay designs where reagent volumes must be tightly controlled. Meridian has developed an active immunoassay blocker, called TRU Block ™ , removes HA, HAMA and RF interference using a completely new approach. TRU Block ™ ™ contains a specialized binder that is directed against antibody interference and is able to bind to HAMA and all types of HA and RF with high affinity. Once TRU Block ™ is bound to the assay interference, it prevents the interference from binding to other antibodies in the assay through
Relative Reduction in HAMA Interference in CA19-9 Assay
4.5
Starting HAMA signal
4
3.5
3
2.5
2
1.5
1 0.5 0
No Blocker
TRUBlock
Competitor
Mouse IgG
No Blocker
5 µ g/mL
2.5 µ g/mL
1.25 µ g/mL
0.625 µ g/mL
Figure 1: A double mouse monoclonal sandwich CA19-9 assay was used to measure the effectiveness of TRU Block ™ , Mouse IgG and a competitor HA blocker in blocking HAMA interference from a commercially sourced patient sample (SD386-15). HAMA activity was measured in the absence (no blocker) and in the presence of blockers (TRU Block ™ , Competitor, Mouse IgG) at various concentrations (0.625 µ g/ml, 1.25 µ g/ml, 2.5 µ g/ml and 5 µ g/ml). Greater reduction in the HAMA signal compared to the no-blocker control (blue) indicates higher HAMA blocking effectiveness.
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Performance Comparison – TRU Block ™ vs Competitors (Patient Sample: HAMA Serum 61)
Relative Reduction in RF Interference in CA19-9 Assay
0 0.2 0.4 0.6 0.8 1 1.2
3,5
3,0
2,5
2,0
1,5
1,0
0,5
No Blocker
TRUBlock
Competitor
Mouse IgG
Rabbit IgG
0,0
No Blocker
5µg/mL
2.5µg/mL
1.25µg/mL
Competitor A
Competitor B
TRU Block TM
Figure 2: A double mouse monoclonal sandwich CA19-9 assay was used to measure the effectiveness of TRU Block TM , Mouse IgG and a competitor HA blocker in blocking RF interference from a commercially sourced patient sample (A12916H). RF activity was measured in the absence (no blocker) and in the presence of blockers TRU Block TM , Competitor, Mouse IgG and Rabbit IgG) at various concentrations (1.25 mg/ml, 2.5 mg/ml and 5 mg/ml). Greater suppression of the RF signal with no blocker (blue) indicates greater RF blocking effectiveness.
(Patient Sample: HAMA Serum 69)
3,5
3,0
2,5
monoclonal ELISAs with blockers added to the sample diluent buffer. The results indicate that TRU Block TM can outperform both Mouse IgG and the other blocker in ELISA-based immunoassays. TRU Block TM has also been successfully used in chemiluminescent assays and lateral flow rapid tests where TRU Block TM was dried down as a stripe between the sample pad and assay antibody location.
2,0
a steric hindrance mechanism. Performance advantages of TRU Block TM include broader coverage against all types of HA inference and RF, the ability to be used at a low concentration, and better blocking efficacy compared to Mouse IgG. The blocking effectiveness of TRU Block TM has been evaluated against Mouse IgG and a well-known HA blocker
1,5
1,0
0,5
0,0
Competitor A
Competitor B
TRU Block TM
(Patient Sample: HAMA Serum 70)
using both two-step (Figure 1) and one-step (Figure 2) double mouse SUMMARY
3,5
3,0
2,5
2,0
Heterophilic antibody interference blockers are an essential part of clinical diagnostic assays, and their importance has been documented by dozens of case studies where approved assays resulted in misdiagnosis (Bolstad, N. et al, 2013). In selecting a blocker, it is important to consider the source of the antibodies used in the assay and the types of heterophilic interference that could impact the assay. TRU Block ™ is a unique HA, HAMA and RF interference blocker that can be used in ELISA, LF and chemiluminescent assay formats. It has several performance advantages over other heterophilic antibody interference blockers on the market and when used in an immunoassay, can prevent false positive and false negative results. Bolstad, N., Warren, D. J. & Nustad, K. Heterophilic antibody interference in immunometric assays. Best Pract Res Clin Endocrinol Metab 27, 647–661 (2013).
1,5
1,0
0,5
0,0
Competitor A
Competitor B
TRU Block TM
250 µ g/mL
125 µ g/mL
62.5 µ g/mL
31.25 µ g/mL
No blocker
Figure 3: Customer (IVD manufacturer) One-step ELISA results: HAMA interference for three different samples (HAMA Serum 61, Hama Serum 69 and HAMA Serum 70) was measured initially in the absence of blockers to determine 100% interference signal level. HAMA activity was then measured in the presence of blockers (added with assay antibodies together) to measure the suppression of signal. Greater suppression of signal (reduced bar height) indicates a more effective HAMA blocker.
Product
Cat Number Protein Concentration
Application
TRU Block TM
A66800H Range: 24 - 26 mg/mL
ELISA
TRU Block TM 2
A66802H Range: 24 - 26 mg/mL
ELISA, CLIA & LF
TRU Block TM 3
A66803H 24.3 mg/mL
ELISA, CLIA
Ordering information: USA
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