## Appendix A TFL-006 immunostaining of the two beadsets
### Conclusions  
The objective of an immunoassay is to monitor the MFI of the sera Abs against a target antigen coated on a solid matrix. In the enzyme-linked immunosorbent assay (ELISA), the binding affinity of the Abs is measured quantitatively as titers, after serially dilutingthe sera. The solid matrix in the Luminex multiplex immunoassay is single-antigen beads. The assay enables the measuring of Abs against a hundred HLA antigens simultaneously, by using the antigen-coated beads. Titration can be conducted [51]; however, titration on every serum of SABs coated with the two classes of HLA is cost-prohibitive. Hence, the sera are not diluted serially. Often undiluted serum is used, which results in a \"prozone effect\". In this study, the sera are tested after a 1:10 dilution, and hence the assay remains semi-quantitative. There is a need for a reliable and accurate measurement of the MFI of the antibody. This can be achieved only after a stringent and empirical assessment of the SAB assay protocol. As frequently observed [14; 15; 16; 46; 51; 59], the accuracy may be affected by an admixture of OC with CC on the SAB, as well as by the use of polyclonal Fab as the detection antibody (_IgHPolyFab_). This study indicates that the accuracy in measurement can be attained using SAB coated with HLA CC only, and with Fc-specific monoclonal IgG (_FcMonolgG_). The costs of LS-SAB and LC-SAB do not differ much. However, clinicians in the USA tend to use LS more often than LC, despite several compelling reports [41; 46; 49; 51; 58; 59]. Most strikingly, [PERSON] et al. [41] from Scotland compared the so-called \"prozone effect\" on LS- and LC-SABs, and documented that the \"prozone effect\" was specific for LS-SAB and not observed with LC-SAB. A recent report [58] comparing the FCXMs of sera that are reactive against HLA OCs, with sera that are reactive against only HLA CCs, showed that the sera reacting strongly with LS-SAB, but not with LC-SAB, gave negative T- and B-cell FCXMs. In contrast, the sera that reacted strongly with LC-SAB, but poorly with LS-SAB, exhibited positive FCXMs. The detection of Abs directed against HLA OCs in LS-SAB assays may lead to an inappropriate listing of unacceptable antigens and pre- or post-transplant desensitization procedures. [PERSON] et al. [15] and [PERSON] et al. [49] have emphasized that such HLA antibodies on OC-coated (_\(\beta\)2-microglobulin-free HLA_) SAB can lead to \"dire consequences,\" namely, \"inappropriate assignment of unacceptable antigens during transplant listing and possibly inaccurate identification of DSA post-transplant.\"  
In conclusion, we recommend LC-SAB for monitoring HLA antibodies for end-stage organ disease patients, both before and after transplantation. The sensitivity of detection can be much improved with _FcMonolgG_. In the interest of avoiding the \"dire consequences\" stated above, and to eliminate the \"prozone effect\" once and for all, the vendors should seriously consider manufacturing the HLA-I SAB to be negative for mAb TFL-006, and HLA-II SAB to be non-reactive to mAb FJ5109. The inclusion of _FcMonolgG_ with the vendors' kits would provide extraordinary service and be cost-worthy for the patients. [PERSON] formulated the overall concept and experimental design and supervised all experiments performed by his research assistants at Terasaki Foundation Laboratory. [PERSON] significantly contributed to the development of the idea since 2017, regularly discussed the progress of the experiments, data analysis, and contributed significantly in drafting and critically reviewing the manuscript at every level of its development. [PERSON] carried out cPRA analysis for all the patients and contributed to the sections about cPRA. All authors have read and agreed to the published version of the manuscript. [PERSON] received a research grant from Immunoc Inc. (July 2017-July 2018), while at the Terasaki Foundation Laboratory. [PERSON], the first son of Late [PERSON], provided necessary funds while completing and writing the project.