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Validation and cross-reactivity pattern assessment of monoclonal antibodies used for the screening of donor-specific IgG antibody subclasses in transplant recipients
  • +2
  • Vadim Jucaud,
  • Anh Nguyen,
  • Bach Tran,
  • Judy Hopfield,
  • Tho Pham
Vadim Jucaud
Terasaki Research Institute
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Anh Nguyen
Terasaki Research Institute
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Bach Tran
Terasaki Research Institute
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Judy Hopfield
Terasaki Research Institute
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Tho Pham
Terasaki Research Institute
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Abstract

The screening for IgG subclass donor-specific antibodies (DSAs) in allograft recipients uses IgG1-4 subclass-specific monoclonal antibodies (mAbs) that should be mono-specific. The cross-reactivity discrepancies reported for IgG subclass-specific mAbs warranted a critical cross-reactivity pattern analysis of the IgG subclass-specific mAbs most commonly used to detect DSAs. We tested the reactivity of 2 anti-IgG1-, 3 anti-IgG2-, 1 anti-IgG3-, and 2 anti-IgG4-specific PE-conjugated mAbs against microbeads coated with IgG1-4 proteins separately. Each IgG subclass protein was coated at three densities on the beads (0.5, 1 and 2 µg of protein per 106 beads), and the PE-conjugated mAbs were titrated from 0.04µg/mL to 5µg/mL. The IgG subclass reactivity of the sample was acquired on the Luminex multiplex platform. Among the IgG subclass-specific mAbs, only the anti-IgG3 (clone: HP6050) mAb was mono-specific. All other mAbs tested were binding to IgG subclass proteins other than their respective immunogen, thereby being cross-reactive. IgG subclass cross-reactivity patterns were dependent on the concentration of both IgG subclass-specific mAbs and IgG1-4 protein targets coated onto the beads. With the current IgG subclass mAbs available, 3 of the 15 possible combinations of IgG1-4 subclass protein could be identified. While the remaining 12 unique combinations cannot be distinguished clearly, 6 groups that corresponded to two different unique combinations of IgG1-4 subclass protein could be identified. The dilution of serum samples and IgG subclass-specific mAbs, other than the anti-IgG3 (clone: HP6050), must be further optimized before their implementation of IgG subclass DSA screening in allograft recipients.