## 3 Results
#### 3.1.2 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):  
Group A: The MFI of anti-HLA IgG on both the SABs (HLA-I/ Figure 2; HLA-II/Figure 3); Group B: The MFI of anti-HLA IgG on LS only (HLA-I/ Figure 2; HLA-II/Figure 3); Group C: The MFI of anti-HLA IgG on LS only (HLA-I/ Figure 2; HLA-II/Figure 3)  
All the three groups of IgGs reacting to HLA-I alleles can be found in one patient's serum. For example, the sera IgG of patient 1 DM reacted with 34 HLA-I alleles, of which 17 are located on both LS and LC (Group A), ten are observed on LS only (Group B), and seven are noted with LC only (Group C).  
The following Abs reacted with HLA-I alleles on both the SABs (Figure 2A):  
A*01:01, 02:03, 11:02, 23:01, 24:03, 29:01, and A*80:01 (7 out of 28 HLA-A alleles);  
B*15:12, 40:02, 44:02, 47:01, 49:01, and B*54:01 (6 out of 43 HLA-B alleles);  
C*01:02, 02:02, 03:03, 03:04, 06:02, 08:01, and C*14:02 (6 out of 13 HLA-C alleles).  
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 11 DM 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 13 LE, 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., 3 LE), it is suggested that the auto-Abs may be binding to OC, due to the exposure of shared epitopes.  
Figure 3: Three groups of serum IgG antibodies in ESRD patients reacting to HLA-II antigens on either LS or LC, or to both SABs, when tested with two different detection Abs (_FcMonolgG_ and _IgHpolyFab_). MFI values are expressed as thousands. 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. Sera of patients 2 DM, 6 FG, 10 DM, 12 DM, 19 HN and 20 HN 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.  
Figure 4: Summary of the profiles of MFI of anti-HLA IgG Abs monitored in the sera of ESRD patients with LABScreen (LS) and LIFECODES (LC), and with two different detection Abs (_FcMonolgG_ and _IgHPolyFab_). Note (1) higher sum and mean MFI values for different sensitization groups with _FcMonolgG_ than with _IgHPolyFab_ (by minimum two- to maximum four-fold) and (2) the higher number of folds of increase in sum and mean MFI with LIFECODES compared to LABScreen.  
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.