Luminex Multiplex Bead Assay Monitoring HLA IgG Antibodies in Sensitized Pre- and Post-Transplant Patients: Clonality of the Detection Antibody Impacts Speciﬁcity and Sensitivity

: The number and the binding afﬁnity, measured as the mean ﬂuorescent intensity (MFI) of HLA-speciﬁc IgG antibodies, formed in the sera of end-stage organ disease patients and allograft recipients, referred to as sensitization, may restrict the availability of a donor organ and/or lead to graft failure after transplantation. The MFI of HLA Abs in sera is monitored with the Luminex-based single-antigen bead (SAB) immunoassay. The following two factors may impact the reliable measurement of MFI: one, the HLA structural variants on the SAB, namely, trimeric HLA (closed conformers, CC) and monomeric heavy chains (open conformers, OC); and two, the nature of the detection Abs, namely, IgG heavy-chain binding polyclonal-Fab ( IgHPolyFab ) or Fc-binding monoclonal-IgG ( FcMonoIgG ). Anti-CC Abs correlate with positive ﬂow cross-matches, and are considered to be pathogenic and damaging to the graft, whereas anti-OC Abs appear to have little relevance to graft attrition. The presence of both CC and OC on beads may impair the reliability of monitoring the nature and MFI of pathogenic Abs. Our objective is to compare the MFI of the HLA Abs in the sera of 20 sensitized patients in two different SAB assays, with the two detection Abs. Our data reveal that the admixture of OC with CC on beads will affect the reliability of the measurement of the pathogenic Abs, and that FcMonoIgG is the more sensitive and speciﬁc detection Ab for the accurate assessment of HLA sensitization. mean ﬂuorescent intensity (MFI)


Introduction
The presence of pre-existing anti-HLA antibodies (Abs) is referred to as sensitization. It is one of the significant immunological barriers to organ transplantation. Sensitization in patients waiting for transplantation is known to be 30% of kidney [1,2], 18% of heart [3,4], 29% of lung [5,6], and 20% of liver transplant candidates [7]. The level of sensitization is assessed based on the number of HLA antigens recognized by the serum IgG Abs, and on the MFI of the anti-HLA Abs at a particular dilution. These Abs may (a) restrict the availability of a given organ for a particular patient, and are a significant cause of long-term graft attrition post-transplantation [8][9][10][11][12][13][14][15]; (b) react with mismatched intact HLA antigens on a donor organ (donor-specific Abs, DSA); and (c) recognize non-donor-specific allo-HLA (NDSA). Although DSA are associated with reduced allograft survival [13,15], it is increasingly observed that not all DSA result in graft failure [11,[16][17][18][19][20][21][22]. These observations suggest possible pitfalls in the immunoassay, and raise concerns about the specificity and sensitivity of the assay for the reliable measurement of the levels of HLA Abs.
HLA Abs, associated with pre-and post-transplant sensitization [8][9][10]12,13], are monitored with Luminex-based multiplex single-antigen bead immunoassay (SAB immunoassay), in which SABs are coated with individual HLA molecules. A vast majority of reports have observed HLA Abs using single-antigen SABs (LABScreen, LS) from a vendor. Some of the studies aimed to distinguish Abs binding to HLA-I OC, induced by acid or heat treatment, from those binding to HLA CC (regular beads), which showed that Abs against intact HLA CC, but not those against denatured HLA OC, are predictive of graft failure [11,[16][17][18][19][20][21][22].
Several reports have documented that many DSA monitored with LS-SABs in preor post-transplant sera were not associated with graft failure [11,[16][17][18][19][20][21][22]49,50]. These studies suggest that (1) only specific monitoring of CC-specific Abs would elucidate the causal relationship between the levels of DSAs and NDSAs with graft failure or rejection, and (2) the reactivity of the sera to naturally occurring OC that are admixed with CC in SAB assay may result in the denial of an otherwise compatible organ, or may result in inappropriate pre-and post-transplantation therapies. Therefore, there is a need to monitor HLA Ab binding to CC, without the interference of OC, during the sensitization of end-stage organ disease patients.
Another salient factor of Ab, which may impact the precise measurement of the MFI or density of serum HLA Abs, is the clonality of the detection Abs [51]. The most commonly used detection antibody in SAB immunoassay is IgHPolyFab [51], which is recommended by histopathologists for better resolution and localization of the surface antigens on cells and tissue sections, due to the signal amplification caused by the IgG heavychain binding polyclonal Fab fragments, binding to an antigen-bound primary Ab (Cusabio Catalog, Cusabio Product Center. Introductory Notes for Secondary Antibodies. Available at: http://www.cusabio.com/catalog-21-1.html, accessed on 12 October 2015.). For the detection of IgG subclasses, Fc-specific monoclonal IgG (FcMonoIgG) is used as a detection Ab [52]. For the detection of anti-HLA IgG, FcMonoIgG has not been used to compare or validate the sensitivity and reliability of the different detection Abs for monitoring Abs in the Luminex SAB assay. We have documented that FcMonoIgG may provide a better quantitative assessment (MFI or titer) of anti-HLA IgG measurements, as it binds to a single Fc domain of the primary anti-HLA IgG, in a one-to-one ratio [46,51]. The studies further revealed that when the serum Ab density was low, the binding of IgHPolyFab was amplified. When the serum density was high, the binding of IgHPolyFab diminished. No such variations were observed with FcMonoIgG [48]. However, it remains to be elucidated whether the nature (mono-versus poly-clonality, and whole IgG versus Fab fragments) of the detection Abs impacts the assessment of the sensitization of patients before transplantation.
These reports led to the following hypotheses: (1) the number and the MFI of anti-HLA CC Abs may differ due to the presence or absence of OC on the SAB, and (2) the sensitivity of the assay may differ depending on the clonality of the detection Abs, i.e., IgHPolyFab and FcMonoIgG. The present investigation is aimed at testing these hypotheses in a clinically defined sensitized ESRD patient cohort waiting for donor organs.

Patients Sera
Both hypotheses were validated on the sera of 20 sensitized ESRD patients (16 females and four males). The possible causes of sensitization vary among these patients. Ten patients (2DM, 6FG, 7HN, 8DM, 10DM, 12DM, 13LE, 15NA, 16HN and 20HN) were possibly sensitized by pregnancy, five (1DM, 3LE, 11DM, 18PK and 19HN) by transfusion, three (9NA, 14HN, 17LE) due to prior transplantation and two (4LE and 5DM) for reasons unknown. All these patients were waiting for donor organs. The patients 1DM, 3LE, 4LE, and 7HN were transplanted within a year after sample collection for this study. The nature of ESRD is provided in the legend for Figure 1. Sera were randomly selected from the Downstate pre-transplant clinic at Downstate Health Sciences University, University Hospital of Brooklyn (UHB) by Prof. Dr. Allen Norin and Dr. Ballabh Das. SAB/DSA study protocols were reviewed and approved by the Downstate Institutional Review Board (IRB), (IRB #1232938-3 and #341403-1). Sera were examined with LS-and LC-SABs using IgHPolyFab and FcMonoIgG. All assays were carried out simultaneously on a single day by one HLA technologist (Mr. Vadim Jucaud) at Terasaki Foundation Laboratory (TFL).
The panel of molecules coated on LS-SAB and LC-SAB can be common to both LC-SAB and LS-SAB or "unique" to either SAB. The number of common antigens are as follows: HLA-A (n = 28), HLA-B (n = 43), HLA-C (n = 13), HLA-DR (n = 32), HLA-DQA1/DQB1 (n = 17) and HLA-DPA1/DPB1 (n = 13). The pane of molecules unique to each SAB and their Ab reactivities are not included in this report.   [53,54]. To verify the specificity of the mAb TFL-006 and other mAbs generated against HLA-E, they were tested using LS-SAB coated with 31 HLA-A, 50 HLA-B, 16 HLA-C, one HLA-G, two HLA-E, and one HLA-F antigen. The mAbs were categorized into eight groups based on their affinity. Group 1 constituted the HLA-E monospecific mAbs that did not react with any HLA class I antigens other than HLA-E. Group 8 (which included TFL-006 and TFL-007, previously called PTER-006/007) reacted with all of the abovementioned HLA-I antigen-coated beads on the LS-SAB (U.S patent 10,800,847; 13 October 2020). The epitope specificity of the polyreactive anti-HLA-E mAbs was monitored by dosimetric inhibition studies using several synthetic peptides, as reported earlier [55,56]. The binding of mAbs TFL-006 and TFL-007 to HLA-I antigens was inhibited by a synthetic peptide 117 AYDGKDY 123 shared by all alleles of HLA class I loci [55,56], but remain cryptic in native HLA-I as they are masked by β2m (Table 4 in [51]). TFL-006 was tested on LS-SAB (Lots # 10 and 11) and on LC-SAB (lot # 3005613) at different concentrations ranging from 10 to 40 µg/mL. The diluents used are the same as reported [45,46].

The HLA-II Polyreactive mAb TFL-FJ5109 Identifies HLA-II-OC on LS-SAB
The presence of the OC among HLA-II antigens was examined with an HLA-II polyreactive mAb.FJ5109, (source: One Lambda, Canoga Park, CA, USA). The mAb was used on LS-SAB (HLA-II LS2A01009, lot 9) after passing through a protein G column. Due to the restricted availability of the mAb, we have tested LS-SAB only.

Modifications in the Assay Protocol for Comparing the Different SABs
To remove the protocol differences between the vendors as a confounding variable, we used the LS vendor protocol (using 2 µL of beads instead of 5 µL/test, a standard procedure at Terasaki Foundation Laboratory (TFL) and Terasaki Research Institute (TRI)). To keep the number of beads consistent, the LC-SABs were spun down to remove 835 µL of the fluid volume from the stock to concentrate their beads (the initial volume of LC beads is 960 µL). The final volume of the SABs from both vendors is therefore maintained at 125 µL. Our standard operating procedure (SOP) was extensively used at TFL and we had obtained concordant results when performing proficiency testing for the UCLA serum exchange program. While designing the assay, we kept the antibody-to-bead ratio consistent between the vendors. For the assay, neat sera were avoided. The sera (20 µL) were diluted (1/10) and then incubated with 2 µL of beads (for both LS and LC) for 30 min at room temperature (RT) on a shaker. The beads were then washed (3×) with LS wash buffer. The Ab binding to beads was assessed with two PE-conjugated secondary Abs (see below) by incubating the detection Ab (50 µL at 5 µg/mL) for 30 min at RT on a shaker. After washing, the beads were suspended in 1× PBS before acquisition on the Luminex platform. At least 100 beads were counted for each antigen for the SAB from different vendors. On the rare occasion that any variation occurred in the bead count, it was <5%. The assay includes a positive control bead (coated with human IgG) and a negative control bead (no antigen). In addition, we have used negative control serum (demonstrated by other methods considered to be free of anti-HLA IgG) as well as positive control serum, prepared by the vendor by pooling sera from several individuals carrying anti-HLA IgG.
In this investigation, we have recorded MFI after normalizing the trimmed mean MFI. Since we are comparing MFIs of different SABs with potentially differing composition panels (LS with CC admixed with OC and LC with CC only), we normalized as follows: Step 1. We subtracted MFI of PBS only from trimmed MFI. This is an essential step because when antibodies were tested on beads with PBS alone, differential MFI values were observed with different HLA molecules.
Step 2. We used (trim mean MFI-PBS MFI) to subtract the following: A. Negative control MFI (NC MFI); B. Negative sera value obtained with LS-SABs (NGS-LS); C. Negative sera value obtained with LC-SABs (NGS-LC); D. B and C were added and divided by 2 for correction.
The following formula explains the steps used: These steps are critical for comparing the two different kinds of SABs and very useful for considering the diverse composition of the panels and the different frequencies of the target epitopes on the beads. In the above protocol, no background noise was observed for any secondary antibody, whether IgHPolyFab or FcMonoIgG. Note that the protocol considers, while calculating normalized trimmed means, the factors that may cause background noise, such as PBS alone or negative controls.

The Diversity of the Secondary Detection Abs
The rationale for comparing two different detection Abs was elaborated in earlier reports [48,52]. The following two PE-conjugated detection Abs were used: IgHPolyFab: PE-conjugated human IgG heavy chain (IgH)-specific polyclonal goat anti-Human IgG Ab fragments [F(ab) 2 ], supplied as 0.5 mg/mL in PBS pH 7.6, by One Lambda Inc (Canoga Park, CA) Cat # LS-A82. The label on the box of the vial (the product literature) states that the product is "PE-conjugated Goat-anti-Human IgG; R-phycoerythrin-coated AffiniPure F(ab) 2 ; goat X human IgG 1 mL (100×)".
FcMonoIgG: PE-conjugated human IgG Fc-specific mouse monoclonal IgG, a whole purified IgG, supplied as 0.5 mg of purified IgG in 1 mL of borate-buffered saline (pH 8.2) by Southern Biotech (Birmingham, AL Cat # 9040-09). The product insert states that the subtype of the IgG is IgG1k, derived from clone JDC-10 and reacts specifically with human/rhesus/chimpanzee IgG Fc (MW 150 kDa). FcPolyIgG, recommended by LC vendor, was not used for the reasons reported in our earlier report [51].
All anti-HLA Ab analyses for HLA-I and -II were performed by the same TFL HLA technologist (VJ) on the same day and the same run on a single tray. We performed experiments using sera diluted 1:10 to avoid falsely low values in the case of antibody excess (the "prozone effect"). The binding affinity of any detected Ab as measured by normalized MFI was compared, using an MFI cut-off for positivity of 500, at serum dilution 1:10. The MFI cut-off was determined for each SAB with each detection Ab based on our earlier report [51] after studying their titration profiles.

cPRA Calculations
Panel-reactive antibodies (PRA) values have been the measurement of preformed HLA Abs (sensitization), which are considered as a formidable barrier to transplantation [57]. United Network for Organ Sharing (UNOS) implemented a strategy using unacceptable HLA antigens and a calculated PRA (cPRA) to award sensitization points. For example, a patient with 80% cPRA would be crossmatched incompatible with 80% of donors. The MFI obtained with Luminex SAB assay is utilized for calculating % of cPRA. For this experimental investigation, the cPRAs for each patient were calculated for each vendor's SAB with each of the detection Abs, using the cPRA calculator on the UNOS website (https: //optn.transplant.hrsa.gov/resources/allocation-calculators/cpra-calculator, accessed on 30 May 2021).

Statistical Analysis
Jeff Gornbein of UCLA/SBCC performed the statistical analyses. Paired comparisons of the number of alleles recognized and the antibody MFI responses were made between LC-SAB and LS-SAB for FcMonoIgG and IgHPolyFab, as well as between FcMonoIgG and IgHPolyFab for LC-SAB and for LS. Since the MFI data did not follow a normal (Gaussian) distribution, the non-parametric p-value for these comparisons was computed using the Wilcoxon signed-rank test for paired data.  (I) The number of HLA-I and HLA-II alleles recognized by sera Abs is significantly higher on the LS-SAB than on LC-SAB, with both the detection Abs (numbers in bold in Figure 1); (II) The FcMonoIgG recognized a significantly (two-tailed t-test) higher number of Abs than IgHPolyFab, for both the HLA-I and II antigens (number in bold in Figure 2), from moderate-to-high sensitization groups; (III) Sera IgG of several patients (2DM, 6FG, 10DM, 12DM, 19HN, and 20HN) did not react with any of the HLA-II antigens, with both the detection-Abs, while these sera reacted moderately (2DM and 6FG) or highly (10DM, 12DM, 19HN and 20 HN) with HLA-I antigens.
Inferences: (1) More HLA antigens are recognized in LS-than in LC-SABs. (2) In moderately or highly sensitized groups, FcMonoIgG recognized more HLA alleles than IgHPolyFab, for both HLA-I and HLA-II. (3) The HLA sensitization in several ESRD patients may differ between HLA-I and HLA-II antigens.   Group A: Abs binding to both SABs; Group B: Abs binding to LS-SAB only; Group C: Abs binding to LC-SAB only. IgG antibodies recognizing antigens unique to the SABs are excluded. The sera were tested at 1/10 dilution and the cut-off of the normalized/trimmed mean MFI is 500. Sera of patient 8DM showed no anti-HLA-I Ab reactivity. HLA antigen with @ sign refer to patient's self-antigen recognized by the patient's IgG Ab. *** Two different kinds of Ab reactivity against a particular allele. One kind of Ab binds LS only (B) and another kind binds to LC only (C), suggesting that it may be due Abs binding LS only (B) may be binding to the heavy chain only or open conformers, OC) and another class of Ab binding LC only(C) may be binding to intact HLA (closed conformers, CC). MFI values are indicated in numbes, each number refering to thousands. For example 10 implies ten thousand and 10+ implies 10,500-10,999.

Differential Binding Patterns of anti-HLA IgG Abs on LS-and LC-SABs
The MFI of sera HLA Abs tested at a 1:10 dilution can be categorized into the following three groups based on the differential binding to LS and LC (Figures 2 and 3): Similarly, all three groups of IgGs reacting to HLA-II alleles can be found in one patient's serum (Figure 3). For example, the sera Abs of 11DM responded with 10 HLA-II alleles on both LS and LC (Group A), six alleles on LS only (Group B), and three alleles on LC only (Group C). In patient 13LE, the serum IgG reacted with 11 HLA-II alleles on both LS and LC (Group A), six alleles on LS only (Group B), and three alleles on LC only (Group CI).
Some serum Abs are directed against self-HLA antigens (with a low MFI). They include HLA-A, HLA-B, and HLA-DRB alleles, and are most often on LS beads (these antigens and their respective MFIs are marked @ in the row of the alleles in Figures 3 and 4). Since such auto-Abs are rarely observed on LC-SAB (e.g., 3LE), it is suggested that the auto-Abs may be binding to OC, due to the exposure of shared epitopes.   showed no anti-HLA-II Ab reactivity. The sera were tested at 1/10 dilution and the cut-off of the normalized/trimmed mean MFI is 500. HLA antigen with @ sign refer to patient's self-antigen recognized by the patient's IgG Ab. *** Two different kinds of Ab reactivity against a particular allele. One kind of Ab binds LS only (B) and another kind binds to LC only (C), suggesting that it may be due Abs binding LS only (B) may be binding to the heavy chain only or open conformers, OC) and another class of Ab binding LC only(C) may be binding to intact HLA (closed conformers, CC). MFI values are indicated in numbes, each number refering to thousands. For example 10 implies ten thousand and 10+ implies 10,500-10,999.
Appl. Sci. 2021   Inference: The sera IgG reacting to the HLA alleles on LS failed to recognize the same molecules on LC, suggesting that these antibodies are bound to the OCs on LS. The sera IgG reacting to the HLA alleles on LC were unable to recognize the same molecules on LS, implying that the OC on LS may interfere with, or cause steric hindrance to, the binding with the CC on LS.

Differences in the Resolution with and the Sensitivity of the Detection Abs
The MFI levels of the sera Abs tested at a particular dilution (1:10) measure the binding affinity of the anti-HLA Abs. The MFI of the anti-HLA Abs that are reactive on both the SABs (Figure 2) also differs with different detection Abs. The MFI values obtained using FC-binding monoclonal IgG are consistently higher than the MFI obtained with IgG heavy chain-binding polyclonal Fab fragments. a.
The MFI obtained for anti-HLA Abs against HLA-I alleles are frequently higher with FcMonoIgG; b.
In addition to HLA antigens that are common to both SABs, few antigens are unique to either LS or LC (data not presented).  Figure 2A). Similarly, the positive MFIs for Abs against HLA-II, with a IgHPolyFab concomitant with low or negative values, were observed with FcMonoIgG on LS-SAB for DQB1*04:02\DQA1*02:01 (e.g., sera # 5F47, 9M33 and 15F43).
Inference: These exceptions observed on LS-SAB could be due to the presence of an OC on the LS, and a lack of the same in LC. Figure 4 summarizes the disparity based on the total sum and mean MFI of IgGs reacting to HLA-I antigens in the four different categories of tests (LC and FcMonoIgG; LC and IgHPolyFab; LS and FcMonoIgG; and LS and IgHPolyFab).
(a) Both the sum and mean MFI values of the anti-HLA-I Abs in group 2 and 3 (moderately or highly sensitized groups) are persistently higher with LC than with LS when tested with FcMonoIgG. All three groups combined are strikingly higher with LC than with LS when tested with FcMonoIgG; (b) With LC-SAB, the sum and mean MFI values for HLA-I Abs assessed using FcMonoIgG are 4.4-fold higher than with IgHPolyFab; whereas, with LS-SAB, the reactivity with FcMonoIgG is just 2.2 to 2.4 higher than that of IgHPolyFab; (c) In poorly sensitized patients, the sum and mean MFIs are higher with LS than with LC when tested with FcMonoIgG or IgHPolyFab. The MFIs with both the SABs with IgHPolyFab, though almost half of the values were obtained with FcMonoIgG, were higher with LS than with LC; (d) Similarly, both the sum and mean MFIs of all anti-HLA-II reactivity with FcMonoIgG are significantly enhanced four-fold, relative to IgHPolyFab; (e) The maximum MFI observed with FcMonoIgG, compared to IgHPolyFab, is higher for LS than for LC.
Inference: It is evident that FcMonoIgG enhances the sensitivity of the SAB immunoassay, particularly while monitoring anti-HLA Abs in the sera of highly sensitized patients. The higher sum and mean MFI values with LS could be due to the presence of an OC.

Documentation of the Open Conformers (OC) on the Different Lots of LS and Lack of the Same in LC-SAB
The presence of an OC on LS-SAB, and the absence of the same on LC-SAB, were shown using the monoclonal antibody TFL-006, which binds to the OC of all HLA class I molecules [45,46]. The presence of lot-to-lot variations in a vendor SAB is known, as every vendor may change the profile of the HLA coated on new lots. In the present study, the lots of SABs used are different from those used in previous reports, and hence they were assessed for the presence or absence of an OC using TFL-006. Figure 5A shows that mAb TFL-006 did not bind to HLA-A (n = 31 alleles), HLA-B (n = 50), and HLA-C (n = 16) molecules on the lot of LC-SABs (lot 3005613), even at 20 µg/mL, while the mAb showed positivity for all the HLA-I antigens coated on the two lots of LS-SABs (#10 and #11).
Inference: The findings confirm that LS carries admixtures of HLA-I CC with OC, while LC only has HLA-I CC.
Similarly, mAb FJ5109 may bind to several HLA-II OC. Figure

Documentation of the Open Conformers (OC) on the Different Lots of LS and Lack of the Same in LC-SAB
The presence of an OC on LS-SAB, and the absence of the same on LC-SAB, were shown using the monoclonal antibody TFL-006, which binds to the OC of all HLA class I molecules [45,46]. The presence of lot-to-lot variations in a vendor SAB is known, as every vendor may change the profile of the HLA coated on new lots. In the present study, the lots of SABs used are different from those used in previous reports, and hence they were assessed for the presence or absence of an OC using TFL-006. Figure 5A shows that mAb TFL-006 did not bind to HLA-A (n=31 alleles), HLA-B (n=50), and HLA-C (n=16) molecules on the lot of LC-SABs (lot 3005613), even at 20 μg/mL, while the mAb showed positivity for all the HLA-I antigens coated on the two lots of LS-SABs (#10 and #11).
Inference: The findings confirm that LS carries admixtures of HLA-I CC with OC, while LC only has HLA-I CC.
Similarly, mAb FJ5109 may bind to several HLA-II OC. Figure 5B    . The binding affinity of antibodies is measured as normalized MFI on the SABs with the mAb. Since the mAb binds to all of the three loci of HLA-II molecules, presence of a shared epitopes among all HLA-II molecules is indicated.    Tables 3 ** see Grop HLA-II details in Tables 4: Table 1 provides details of patient ID HLA-I HLA-II Sensitization Groups* as in Table 2 Serum ID*** % cPRA & Median MFI

LIFECODE (LC) LABScreen (LS) Sensitization
Groups** as in Table 2 Serum  (b) The % cPRA may not be reliable with LS, possibly due to the presence of an admixture of OC with CC, due to the negative cPRA observed with LC in the following patient: 5DM.
Based on the negative (total zero) cPRA obtained with LS using IgHPolyFab, the patients could have transplanted an organ without recognizing the presence of unacceptable antigens. This can be avoided by using FcMonoIgG with LC-SABs. In contrast, an inaccurately inflated cPRA, due to the OC, could result in declined offers for potentially compatible organs in high % cPRA patients.
Inference: HLA Abs monitored on LC using FcMonoIgG are more reliable for monitoring cPRA than the conventional protocol of using LS with IgHPolyFab. , and the reactivity is significantly higher with LC than with LS. IgHPolyFab showed low or no binding with both the SABs. Evidently, LC-SABs with FcMonoIgG as the detection Ab are reliable for monitoring intact HLA or CC. FcMonoIgG provides greater sensitivity and specificity (resolution) than IgHPolyFab, with a more accurate estimation of binding affinity (MFI). This is important because the Luminex SAB assay is semi-quantitative. Our present and previous results [46] caution that LS-SAB and polyclonal detection Abs should be avoided for calculating % cPRA. If not, potentially compatible organs may be denied, or inappropriate administration of costly and possibly toxic desensitization procedures may result.
Several HLA alleles are recognized by the patients' sera on CC-specific LC-SABs, but not on LS-SABs (Figure 7). The failure of these CC-specific IgG Abs to recognize LS-SABs could be either due to (1) the low density of CC on LS-SAB, or (2) the steric hindrance caused by the admixture of OC with CC on LS. Figure 7 also highlights a unique profile in that several sera IgGs bind only with LS-SABs, with both IgHPolyFab and FcMonoIgG. The failure of these IgG Abs to recognize LC-SABs could be due to the lack of OC on LC. Those IgG binding only to LS are likely to be OC-specific IgG Abs. These findings explain why not all donor-specific Abs (DSAs) observed with LS-SABs result in graft failure [11,[16][17][18][19][20][21][22]42,43].
It is known that the results of SAB assays often fail to correlate with the results of FCXM tests. Recently, we have reported (data submitted for publication [58]) that sera reacted strongly with LS-SABs (>13,000 MFI), but weakly or not at all with LC-SABs (<1000 MFI), which gave negative T-and B-cell FCXMs [58]. In contrast, sera that reacted with LC-SABs (>13,000 MFI), but weakly with LS-SAB (<2100 MFI), exhibited positive FCXMs [58]. The detection of Abs directed against the OC in SAB assays may lead to an inappropriate listing of unacceptable antigens, pre-or post-transplant desensitization procedures, or the decision not to XM.
The findings reported in this investigation gain support from the works of Gao et al. [14] and Porcheray et al. [59], who have developed a unique B-cell clone (4G10) from an explanted kidney graft of a transplant recipient. The reactivity of the clone supernatants and the patient's serum to HLA-I were assessed using regular LS-SABs, iBeads (enzymatically treated LS-SAB to remove OC-and CC-specific), and the acid-treated LS (denatured OC). The mAb 4G10 showed no reactivity to iBeads, but reacted with a majority of B and C locus antigens on regular LS-SAB and acid-treated LS, confirming the affinity of the mAb 4G10 for OC on the LS. Notably, both mAb 4G10 and the patient's sera were negative by complement-dependent crossmatch (CDCXM) testing, suggesting the lack of pathogenic relevance of anti-OC Abs.  It is known that the results of SAB assays often fail to correlate with the results of FCXM tests. Recently, we have reported (data submitted for publication [58]) that sera reacted strongly with LS-SABs (>13,000 MFI), but weakly or not at all with LC-SABs (<1000 MFI), which gave negative T-and B-cell FCXMs [58]. In contrast, sera that reacted with LC-SABs (>13,000 MFI), but weakly with LS-SAB (<2100 MFI), exhibited positive FCXMs [58]. The detection of Abs directed against the OC in SAB assays may lead to an Similarly, Michel et al. [49] confirmed the presence of OC on LS-SAB, using mAb HC-10. Indeed, no patients in their study had Ab-mediated rejection, and had antibodies reactive to β2m-free HLA (OC). Further, they emphasized that "reactions due to the presence of β(2)-m-fHLA [OC] . . . can lead to the inappropriate assignment of unacceptable antigens during transplant listing and possibly inaccurate identification of DSA in the post-transplant period"(p. 356, [51]).
Susal and co-investigators [15,16] observed that the sera Abs that reacted with LS-SABs were non-reactive with complement-dependent crossmatch (CDCXM) testing. They further examined ten sera of waiting list patients who responded positively for anti-HLA Abs on LS-SAB with LC-SAB. All the ten sera that were positive with LS were reactive to A*24:02, B*08:01, B*15:12, B*44:02, B*37:01 C*05:01, and C*17:01. However, with LC-SAB, eight of the ten patients failed to show HLA Abs reactivity. Of the different HLA Abs detected by LS, only A*24:02, B*08:01, and B*15:12 were detected with LC for the two LC-positive sera. They noted that the sensitized patients that were positive for five alleles with LC were negative with LS-SAB. In conclusion, there was no association of kidney graft loss with the HLA Abs detected exclusively using LS-SAB with IgHPolyFab. They state that "HLA mismatches defined according to the HLA antibody specificities in a potential recipient's serum, the smaller the likelihood that a donor kidney will be considered suitable for this patient. The erroneous assignment of HLA antibodies, and consequently of "unacceptable HLA mismatches, can therefore have dire consequences" (p. 2080, [15]). Despite these reports [14][15][16]46,51,59], and after direct documentation of the presence of an OC on LS-SAB [44,45], many clinical laboratories continue to use LS-SAB to monitor sensitization in end-stage disease patients and post-transplantation.
Similar findings are also observed for HLA-II. An HLA-II polyreactive mAb FJ5109 ( Figure 5B) revealed the OC of HLA-II on the LS-SAB. The existence of an HLA-II OC on LS-SAB was also supported by earlier reports [50,51]. This study documented ( Figure 5B) that the LS-SAB may carry OC of HLA-II. More in-depth studies are needed, using FJ5109 or similar mAbs, as was done for HLA-I. Figure 4 summarizes the entire findings of this investigation. The median or mean of the pooled MFIs of all alleles are persistently higher with FcMonoIgG than with IgHPolyFab, for both LS-and LC-SABs. The MFI values with FcMonoIgG for LC-SAB are significantly greater than those obtained with LS. The above observations, on the clonality of the detection Abs, do not support the contention of Hilton and Parham [35]. They also compared the binding of polyreactive mAbs (MA2.1 and BB7.1) using LS-and LC-SABs. The mAb MA2.1 bound to A*02:01/02/03/B*57:01/B*58:01 (see Figure 1 in [35]), and mAb BB7.1 bound to HLA-B*42:01, B7 and B27. These mAbs reacted well with LS-SABs, but poorly with LC-SAB. Therefore, they attributed "the lower affinity of MA2.1 and BB7.1 for HLA class I allotypes presented on the Gen-Probe (LC) SAB" "is (due to) the lower antigen density present on these beads" (page 215, [35]). Our study clarifies that the lower affinity of these mAbs for LC could be due to the absence of OC on LC-SAB. In several previous reports [46,54,55,60,61], we have documented that the HLA polyreactive monoclonal antibodies (TFL-006 and TFL-007) bind to shared epitopes found on the HLA OC, which are otherwise cryptic due to the dimerization of the OC with β2-microglobulin.
This study and our previous reports [46,51] stress the need to eliminate IgHPolyFab and replace it with FcMonoIgG, for the monitoring HLA Abs on the Luminex-based SAB assay. This should be the standard protocol for monitoring an array of antigens on Luminex multiplex SAB assays.

Limitations of This Investigation
IgM and four subclasses of IgG are found in normal subjects' sera and patients' sera. The OC generated as a consequence of inflammation are immunogenic, for they expose otherwise cryptic epitopes and are capable of eliciting IgM initially. The failure of the detection Abs to bind to LS-SABs could be due to IgM binding to the OC, causing steric hindrance for IgG binding to CC. Selective binding of non-immunoglobulin serum components (proteins, peptide repertoire, chemo-and cytokines, oligosaccharides, saturated and unsaturated fatty acids, and cholesterol) to HLA OC cannot be ruled out. Lower MFIs observed with IgHPolyFab, in contrast to those of FcMonoIgG, could be due to such steric hindrances.

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 diluting the 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 (FcMonoIgG).
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, Battle 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. Susal et al. [15] and Michel et al. [49] have emphasized that such HLA antibodies on OC-coated (β2-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 FcMonoIgG. 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 FcMonoIgG with the vendors' kits would provide extraordinary service and be cost-worthy for the patients.