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Aim: Not all anti-HLA antibodies are detrimental to graft survival, yet it is difficult to pinpoint detrimental anti-HLA antibodies. Here, we provide a tool for isolating anti-HLA antibodies from complex patient sera to enable the functional and phenotypic characterization of these immunoglobulins. Anti-HLA antibodies were isolated from several sensitized patients and characterized for functional activity and antibody classification. Methods: A soluble version of the class II molecule HLA-DR11 was produced in mammalian cells, purified, covalently coupled to a solid support, and used to recover anti-HLA-DR11 antibodies from the sera of sensitized patients. Sera from patients sensitized by a renal graft were compared to sera from patients not sensitized by an organ. Anti-HLA-DR11 antibodies from these patients were quantified, assayed for HLA specificity and cross reactivity, quantified, and isotyped. Results: HLA-DR11immunoaffinity purification yielded antibodies specific for HLA-DR11 while antibodies for HLA-DQ specificities flowed through the column. Serum concentrations of total anti-HLA-DR11 antibodies did not differ between the two groups and ranged from 5.9μg/ml – 467ng/ml. All patients had anti-HLA-DR11 antibodies from every isotype with enrichments in IgM, IgG2, IgG3, IgE and underrepresentation of IgG1 as compared to bulk serum antibodies. IgG2 was more prevalent in patients whose sensitizing event was a renal graft compared to patients not sensitized by an organ. Conclusions: HLA immunoaffinity chromatography facilitated the purification anti-HLA antibodies from patient sera so they could be assayed apart from sera. The resulting data show that there are qualitative but not quantitative differences in anti-HLA antibodies between patients who were sensitized with a renal graft as compared to patients not sensitized with an organ. These data establish HLA immunoaffinity columns as a tool for isolating anti-HLA antibodies so that the nature of these antibodies can be determined. McMurtrey: Pure Protein: Consultant. Buchli: Pure Protein: Employee. Cate: Pure Protein: Employee. Osborn: Pure Protein: Employee. VanGundy: Pure Protein: Employee. Mojsilovic: Pure Protein: Employee. Hildebrand: Pure Protein: Science Med Advisor. [Copyright &y& Elsevier]

Aim: Strong humoral responses to allogeneic HLA preclude organ transplantation, promote hyperacute rejection, and contribute to chronic transplant rejection. Therapies exist to prevent B cells from secreting antibodies, and bulk antibody removal is also an option, but there is no means to specifically deplete antibodies that recognize an allogeneic HLA molecule while leaving humoral immunity largely intact. Here, we tested a sHLA Antibody Removal Column (SHARC) for removing anti-HLA antibodies from liter volumes of patient samples. Methods: Approximately 70 milligrams of the soluble class II molecule HLA-DRB1∗11:01/DRA1∗01:01 (DR11) produced in mammalian cells was covalently coupled to a solid support matrix. The HLA-DR11 matrix was packed into a column for the purpose of removing anti-DR11antibodies from patient plasma. 2.5L of patient plasma containing anti-DR11 antibodies was run over the column at a clinically relevant flow rate of 40ml/min. Fractions of the flow through were taken and anti-HLA-DR11 antibodies were measured using a single antigen bead assay. Results: When patient plasma recognizing multiple HLA was passed over the DR11 matrix, antibodies specific for DR11were removed while antibodies for other class II (DQ) passed through the SHARC intact. Additionally there was no change in bulk antibody concentration after the plasma was passed over the column. DR11 specific antibodies that bound to the column were recovered by elution and retained their reactivity for DR11 as well as other specificities with expected shared cross reactivity. Conclusions: Native class II molecules were coupled to an affinity matrix, antibodies specific for class II HLA were removed from patient plasma, and the depleted antibodies were recovered intact from the column. These data represent a proof of concept for selective antibody desensitization as a potential therapeutic. McMurtrey: Pure Protein: Consultant. Buchli: Pure Protein: Employee. VanGundy: Pure Protein: Employee. Hildebrand: Pure Protein: Science Med Advisor. [Copyright &y& Elsevier]

Aim: Soluble HLA is found normally in all individuals, and the release of soluble HLA (sHLA) from transplanted organs is thought to modulate allogeneic immune responses. It has been difficult to experimentally measure the quantities of donor specific soluble HLA released following solid organ transplantation, and the aim of this study was to carefully measure total and donor-specific sHLA in serum. Methods: An ELISA with mAb W6/32 and BB7.2 was used to measure total sHLA and sHLA-A2, respectively. Eighty-five healthy controls were studied for total serum sHLA. Twelve patients where neither the donor nor recipient expressed A2 were used to derive a negative threshold for the assay. Twelve cases where the transplanted kidney expressed A2 and where the recipient had antibodies to A2 were studied. Pre-transplant, day 3, 6, 10 & 30 post transplant serum were tested for sHLA-A2. Results: We found the median concentration of sHLA class I in healthy people is 1.6μg/ml ranging from 0.4-6.3. A single sHLA allomorph (sHLA-A2) accounted for about 4% of the total sHLA observed in serum. In transplant recipients, total sHLA concentration at every time point post transplant was significantly lower than healthy controls. Further, sHLA concentrations slowly decreased after transplant from 1.3μg/ml on day 3 to 0.7μg/ml on day 30. Donor specific sHLA (sHLA-A2) was observed in 58% cases, as early as day 6 post transplantation. However the presence of the donor sHLA did not associate with acute antibody mediated rejection (6/7 cases with sHLA had acute AMR, 3/5 cases with no sHLA had acute AMR). Conclusions: Soluble HLA was found at appreciable levels in transplant recipients as well as healthy individuals. In transplant recipients, sHLA concentration decreased over time to a level that is approximately 2x less than normal by day 30. It was also shown that donor specific sHLA in patient sera is detectible in the majority of renal transplants although the presence of donor-derived sHLA did not associate with acute AMR. [Copyright &y& Elsevier]

Aim Some earlier studies indicated that monoclonal Abs can be neutralized and inhibition measured with a bioassay design structured to reduce 95–100% of the maximal signal at 100 μ g/ml sHLA if operated in the mid linear range of the response signal. The aim of this investigation was to determine whether the theoretical concepts describing the basic nature of neutralization of HLA antibodies could be applied to more complex serological specimen resulting in clinically useful information. Methods Sera specimens were tested by individually adding different combinations of sHLA molecules as neutralizing agents prior to response measurement. After subtraction of the resulting inhibitory profile from the non-treated reference profile, a neutralization profile was generated, visualizing Ab responses directly related to the applied neutralizer allele. Results As shown, sHLA-A ∗ 02:01 was able to 100% neutralize A2-related responses, leaving other HLA/antibody interactions untouched. In this example, when also subtracting B ∗ 08:01 and B ∗ 49:01, a near complete inhibition of the reference profile was achieved. Minor assay-to-assay background fluctuations of 10% can generally be observed. Significantly, HLA antigens considered directly responsible for the immunizing event generally showed a complete inhibition of the reference profile, whereas partial inhibitions were seen as indicator of HLA antibody species present with different antigen/epitope correlation. Conclusion Creating a neutralizer profile subtracting the inhibitory profile from the reference profile is most helpful for better analytical assessment of signal patterns, delivering a positive view of the blocking event, ultimately allowing better interpretation of antibody populations. As demonstrated, complex reactivity patterns could be reduced to more simplifying outputs. We believe that reductive profiling techniques will greatly assist in obtaining more accurate and meaningful data in the assessment of risk for immunologic rejection. [ABSTRACT FROM AUTHOR]

Aim The measurement of rejection-causing HLA Abs in serological samples of HLA sensitized individuals has clinical relevance in the evaluation of pre- and post-transplant patients. However, due to the increasing demand for more accurate and meaningful data, the determination of MFI values alone is not sufficient anymore to evaluate a patient’s risk potential. In this study, we aimed to demonstrate the enormous complexity of Ab patterns in serological specimen. Methods To visualize variations and different levels of HLA Ab patterns, several investigative tools were applied including HLA Ab-detection assays, neutralization experiments, sera titration analysis and comparative information from human monoclonal Ab profiles derived from single B cells. Results In tier 1, neutralization experiments clearly demonstrated that original response patterns could be deconvoluted into individual sub-patterns and single alleles identified as primary cause of an immunizing event. Depending on exposure, one or several antigens were generally involved. Tier 2 patterns were originated from a single epitope harbored by the immunizing antigen of origin that naturally cross-reacts with related epitope-sharing antigens. Currently, over 100 human monoclonal Ab patterns were collected defining such epitopes and being used to further resolve Tier 1 patterns by a pattern recognition algorithm. Tier 3 involves either individual epitopes shared among related antigens or Abs that recognize a specific HLA/Ab interaction but not necessarily at the same affinity. Tier 4 includes diverse epitope patterns that appear using different isotype-recognizing detection systems. Tier 5 refers to experiments conducted under a time interval, visualizing the appearance/disappearance of different Ab species in time. Conclusion We believe that the knowledge gained about the apparent complexity of specific immune patterns will help us to reach the next chapter of patient care and contribute to a better risk management in Ab-related graft rejection. [ABSTRACT FROM AUTHOR]

Aim: Due to the increasing demand for more accurate and meaningful data in antibody detection assays, which includes quantitative measures of antibody titers, we evaluated a series of serological specimens to demonstrate the benefit of antibody titration analysis. We show that by identifying the linear range of the assay, semi-quantitative observations are possible where titer comparisons can be easily made and saturation issues such as “prozon effects” are eliminated. Methods: To determine linear range and half maximal effective concentrations (EC50), serological specimens from highly sensitized patients were diluted to eight different concentrations and their MFI signal determined using an in-house, 120-allele Luminex-based single specificity solid phase assay platform. Results: Our results show that individual, single-allele titration curves can be generated to establish optimal performance range. We found that titration analysis has two major benefits: (1) eliminating serological interferences at high concentrations, and (2) semi-quantitative observations within the linear range. According to our test series, most sera will cause interferences and high background if tested undiluted. Accurate determinations can only be made by applying serial dilutions to reach highest signal-to-background ratios. Conclusions: Since the physiological responses of the adaptive immune system against an HLA target are highly variable and antibody composition is distinct to each individual, accurate determination of specificities, titer and strengths of antibodies has been extremely difficult. Titration analysis offers the possibility to evaluate several of such functional traits of antibodies to any given HLA molecule allowing better interpretation of individual antibody populations in a quantitative matter. This project will likely lead to a better assessment of HLA titers and generate new insights for post-transplant monitoring and early recognition of immunologic rejection. [Copyright &y& Elsevier]

Aim: Antibody concentration, isotype, epitope specificity, cross-reactivity, and the ability to fix complement have all been implicated as factors that contribute to the pathogenicity of anti-HLA antibodies. Since humoral responses are typically polyclonal in nature, multiple epitopes with overlapping cross-reactivities are generally observed. In this study, we demonstrate that applying different combinations of soluble HLA (sHLA) as neutralizing agents, specific individual inhibitory patterns can be obtained resulting in the deconvolution of complex reaction patterns. Methods: A two-tier approach was applied to profile serological specimen from sensitized patients. (1) A sera specimen was titrated to establish the linear range of the sera antibody reactivities. (2) A dilution corresponding to a half-maximal signal output was applied and pure sHLA added to neutralize a given allele pattern. For signal evaluation, an in-house, 120-allele Luminex-based single specificity solid phase assay platform was utilized. Results: A scheme of different HLA combinations was applied for each sera to create individual neutralization patterns. Our results show that complex reactivity patterns can be reduced to more simplified outputs allowing better interpretation of antibody populations. Single neutralization patterns correspond well with individual eplets. Furthermore, this technique was also of use in evaluating false positive/negative assay signals obtained by other assay systems. Conclusions: The power of deconvolution will greatly assist in obtaining more accurate and meaningful data to assess transplant patients and also add new insights into anti-HLA antibody profiling and epitope structure analysis. Further experiments using the titration model determining half maximal effective concentrations (EC50) for sera titers and half maximal inhibitory concentrations (IC50) for HLA inhibition may result in a mathematical engraftment factor applied in the assessment of risk for immunologic rejection. [Copyright &y& Elsevier]

Aim: Single antigen beads (SAB) assays are routinely used to identify anti-HLA antibodies and the level of detection is commonly communicated as mean fluorescence intensity (MFI). Many factors may contribute to an MFI value, including antibody concentration and interference of non-IgG isotypes. Here we purified polyclonal anti-HLA antibodies from sensitized patients and tested the influence of antibody concentration and non-IgG isotypes on MFI. Methods: Using an HLA-DR11 affinity column, we purified anti-HLA-DR11 antibodies from 13 sensitized human sera. The purified anti-HLA antibodies were then tested on a SAB assay using a secondary that only recognizes IgG isotypes. IgG isotypes were separated from IgM and IgA isotypes via size exclusion HPLC. Reduction of multimeric antibodies (IgM, IgA) to monomers was preformed with 100mM DTT at 4°C overnight with a total antibody concentration at 1mg/ml. Results: Serial dilution experiments with affinity purified anti-HLA antibodies demonstrated a strong correlation between antibody concentration and MFI within a single patient. However, between patients, the MFI value did not correlate to an antibody concentration – MFI was not comparable between patients. When IgM and IgA multimers were separated from IgG, a significant increase in MFI was observed compared to the original mix of isotypes. Furthermore, reduction of IgM and IgA into monomers with IgG did not impact MFI values as the monomeric mix provided an MFI matching the IgG monomer only. Conclusions: Multiple factors influence SAB MFI. Here we tested the influence of both antibody concentration and the presence of IgM, and IgA on MFI. We show that MFI correlates with antibody concentration within a particular patient but not between patients. We also show that mixing IgM and IgA multimers (but not monomers) with IgG antibodies interferes with the assay by reducing MFI. Together these data exemplify the complexity of anti-HLA antibodies and factors that influence the SAB that is used to detect them. Cate: Pure Protein: Employee. McMurtrey: Pure Protein: Consultant. Buchli: Pure Protein: Employee. VanGundy: Pure Protein: Employee. Osborn: Pure Protein: Employee. Mojsilovic: Pure Protein: Employee. Hildebrand: Pure Protein: Science Med Advisor. [Copyright &y& Elsevier]