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. 2024 Apr;16(7):135-148.
doi: 10.4155/bio-2023-0191. Epub 2024 Feb 22.

Eliminating drug target interference with specific antibody or its F(ab')2 fragment in the bridging immunogenicity assay

Xiaojie Deng  1 , Yingying Hou  1   2 , Wenyi Yuan  1 , Hongzhou Yang  3 , Ruowen Guo  3 , Tingting Liu  1   2 , Yongzhen Liu  1 , Junjiu Xu  1 , Heng Liu  3 , Likun Gong  1   2   4 , Qiuping Qin  1   2
Affiliations

Eliminating drug target interference with specific antibody or its F(ab')2 fragment in the bridging immunogenicity assay

Xiaojie Deng et al. Bioanalysis. 2024 Apr.

Abstract

Background: DB-1003 is a humanized anti-IgE monoclonal antibody with higher affinity than omalizumab. In the affinity capture elution (ACE)-based bridging electrochemiluminescent immunoassay (ECLIA) for antibodies to DB-1003, monkey serum IgE caused false-positive results. Materials & methods: The target-specific antibody or its F(ab')2 fragment was used to mitigate drug target interference in an ACE-based bridging ECLIA for the detection of anti-DB-1003 antibodies. Results: The sensitivity of the developed assay was at least 100 ng/ml. When the anti-drug antibody concentration was 250 ng/ml, the assay tolerated at least 20.0 μg/ml of the monkey IgE. Conclusion: Incorporating the target-specific antibody or its F(ab')2 fragment can overcome the interference from monkey serum IgE in ACE-based bridging ECLIA for anti-DB-1003 antibody detection.

Keywords: IgE; anti-drug antibody; immunogenicity; mitigation; target interference.

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Conflict of interest statement

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Figures

Figure 1.
Figure 1.. IgE in cynomolgus monkey sera interfered with the bridging electrochemiluminescent immunoassay results.
Seven naïve monkey serum samples were tested with the bridging immunogenicity assay and the test results showed interference signal at different levels. Thereafter, the serum samples were tested for IgE concentrations, and the result showed that the higher the concentrations of IgE in the samples, the higher the interference signals of the bridging immunogenicity assay. ECL: Electrochemiluminescence.
Figure 2.
Figure 2.. The bridging electrochemiluminescent immunoassay binding signals in the Lowcross buffer samples spiked with varying amounts of cynomolgus monkey IgE.
IgE at concentrations of 4.57, 13.7, 41.2, 123, 370, 1111, 3333, 10,000 ng/ml was spiked to Lowcross buffer, respectively. Thereafter, the spiked samples were tested with the bridging immunogenicity assay. The signals were closely dependent on the concentrations of IgE spiked in Lowcross buffer. ECL: Electrochemiluminescence.
Figure 3.
Figure 3.. Mitigation of IgE interference by using omalizumab.
To mitigate IgE interference, different concentrations of omalizumab were tried. The test result showed that for the sample with interference signal of as high as 60,881 (its concentration of IgE was not measured at the early time of the assay development), the interference signal reduced steadily as the concentration of omalizumab increased from 100 to 2000 μg/ml. Nevertheless, even when omalizumab concentration reached 2 mg/ml, the target interference signal was not entirely eliminated. ECL: Electrochemiluminescence.
Figure 4.
Figure 4.. Mitigation of IgE interference by using DB-2002.
To mitigate IgE interference, different concentrations of DB-2002 were used. The test result showed that for the sample with interference signal of as high as 138,927, the interference signal reduced remarkably as the concentration of DB-2002 increased from 25.0 to 200 μg/ml. Noticeably, when the concentration of DB-2002 was 50.0 μg/ml, the target interference signal was entirely eliminated. ECL: Electrochemiluminescence.
Figure 5.
Figure 5.. Schematic representation of the bridging assay incorporating affinity capture elution and a blocking agent.
ADA: Anti-drug antibody; Bio-DB-1003: Biotinylated DB-1003; HAC: CH3COOH; MSD: Meso Scale Discovery; Ru-DB-1003: Ruthenium-labeled DB-1003.
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