Luminex Beads Research Articles

New Therapies for Highly Sensitized Patients on the Waiting List.

Exposure to HLA alloantigens through pregnancy, blood products, and previous transplantations induce powerful immunologic responses that create an immunologic barrier to successful transplantation. This is commonly detected through screening for HLA antibodies using Luminex beads coated with HLA antigens at transplant evaluation. Currently accepted approaches to desensitization include plasmapheresis/low-dose or high-dose intravenous Ig plus anti-CD20. However, these approaches are often unsuccessful because of the inability to remove high titer circulating HLA antibodies and limit rebound responses by long-lived anti-HLA antibody secreting plasma cells (PCs) and memory B cells (B MEM ). This is especially significant for patients with a calculated panel reactive antibody of 99%-100%. Newer desensitization approaches, such as imlifidase (IgG endopeptidase), rapidly inactivate IgG molecules and create an antibody-free zone by cleaving IgG into F(ab'2) and Fc fragments, thus eliminating complement and cell-mediated injury to the graft. This represents an important advancement in desensitization. However, the efficacy of imlifidase is limited by pathogenic antibody rebound, increasing the potential for antibody-mediated rejection. Controlling antibody rebound requires new strategies that address the issues of antibody depletion and inhibition of B MEM and PC responses. This will likely require a combination of agents that effectively and rapidly deplete pathogenic antibodies and prevent immune cell activation pathways responsible for antibody rebound. Here, using anti-IL-6 receptor (tocilizumab) or anti-IL-6 (clazakizumab) could offer long-term control of B MEM and PC donor-specific HLA antibody responses. Agents aimed at eliminating long-lived PCs (anti-CD38 and anti-B-cell maturation antigen×CD3) are likely to benefit highly HLA sensitized patients. Complement inhibitors and novel agents aimed at inhibiting Fc neonatal receptor IgG recycling will be important in desensitization. Administering these agents alone or in combination will advance our ability to effectively desensitize patients and maintain durable suppression post-transplant. After many years of limited options, advanced therapeutics will likely improve efficacy of desensitization and improve access to kidney transplantation for highly HLA sensitized patients.

A novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)-1a and -1b antibodies.

To identify specific human neutrophil antigen (HNA) antibodies, assays using neutrophils such as monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) are recommended. However, these assays are limited by labor-intensive neutrophil preparation and varying antigen expression levels. We evaluated a newly developed immunocomplex capture fluorescence assay (ICFA) for identifying HNA-1 antibodies and compared it to MAIGA and LABScreen Multi (LABM), which utilizes recombinant HNA-coated Luminex beads. For ICFA, HNA-1a or HNA-1b transfected cells replaced neutrophils. Cells incubated with serum were lysed, and immune complexes were captured using five CD16 monoclonal antibody-conjugated Luminex beads. Nine antisera with known specificity and 26 samples suspected of containing HNA antibodies were analyzed by ICFA and MAIGA using neutrophils or transfected cells (ICFA-N or ICFA-T, and MAIGA-N or MAIGA-T, respectively). ICFA-T and MAIGA-N accurately determined the specificity of all antibodies in the nine antiserum samples. The ICFA-T detection limit was 2048-fold for anti-HNA-1a and 256-fold for anti-HNA-1b; the limits of MAIGA-T, MAIGA-N, and LABM were 32-, 4 ~ 64-, and 128-fold for anti-HNA-1a and 64-, 16 ~ 64-, and 32-fold for anti-HNA-1b, respectively. Twelve and 7 of the remaining 26 samples tested negative and positive, respectively, in both ICFA-T and MAIGA-N. Antibody specificity against HNA-1a or HNA-1b determined using ICFA-T agreed with that determined using MAIGA-N and LABM. Another seven samples tested positive in ICFA-T but negative in MAIGA-N. The novel ICFA is highly sensitive and exhibits specificity similar to MAIGA and LABM for detecting HNA-1 antibodies.

Structural and biological insights into outer membrane protein lipotoxin F of Pseudomonas aeruginosa: Implications for vaccine application

Due to the increasing antibiotic resistance of Pseudomonas aeruginosa (PA), an effective vaccine is urgently needed. However, no PA vaccine has been approved to date, and new protective antigens are needed to improve their efficacy. In this study, Luminex beads were used to identify new candidate antigens, after which their crystal structure was determined, and their potential contribution to bacterial pathogenesis was assessed in vitro and in vivo. Notably, a significant antibody response against the outer membrane protein LptF (lipotoxin F) was detected in sera from 409 volunteers. Moreover, vaccination with recombinant LptF conferred effective protection in an acute PA pneumonia model. The crystal structure showed that LptF comprises a 3-stranded β-sheet (β1-β3) and three α-helices (α1-α3) that are organized in an α/β/α/β/α/β pattern, which is structurally homologous to OmpA and related outer membrane proteins. In addition, LptF binds to peptidoglycan in an atypical manner, contributing to the pathogenesis and survival of PA under stress. Our data indicate that LptF is an important virulence factor and thus a promising candidate antigen for PA vaccines.

An optimized procedure for Luminex-based human platelet antigen-specific antibody screening and identification (PakLx assay) with a cost-effective approach and improved sensitivity.

Detection of anti-platelet antibodies is required for the diagnosis of foetal/neonatal alloimmune thrombocytopaenia. The most commonly used methods for anti-platelet antibody detection are the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) and the Luminex bead assay (PakLx). However, for economic reasons, the use of the PakLx assay is limited. In the present study, we evaluated the performance of an optimized protocol based on a half-volume of PakLx reagents. We compared two alternative procedures: one with a half-volume of all components including patient samples, and another based on a half-volume of reagents but a standard volume of patient sample. Our results obtained with a panel of 67 samples demonstrate improved sensitivity when using a standard sample volume. In the event of an inconclusive result with this optimized protocol (e.g., incomplete panel of positive Luminex beads), we recommend testing the sample with an alternative protocol (e.g., MAIPA or the original PakLx protocol).

High throughput Luminex beads based multiplex assay for identification of six major bacterial pathogens of mastitis in dairy animals.

Bovine mastitis is caused by over 150 different microorganisms. Specific identification and quantification of multiple bacteria in a single milk sample becomes essential for rapid intervention. In the present study a Luminex beads based multiplex assay emphasizing on the precise identification of six major bacterial pathogens of mastitis was developed. Assay was developed in two triplex sets, triplex 1 comprised of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis while triplex 2 consisted of Staphylococcus aureus, E. coli and Klebsiella pneumoniae. The analytical sensitivity was 10 6 copies per reaction mixture for all the six bacteria. A 100% analytical specificity was observed for simultaneous detection of these bacteria. Clinical milk samples from 100 bovine quarters were tested for validation. The analytical sensitivity was similar to the findings reported earlier in real time PCR multiplex assay targeting the DNA of the 11 most common bacterial species or groups in mastitis. The analytical specificity of the optimized assay was 100% similar to reported earlier for simultaneous detection of Mycoplasma spp. and for seven entric viruses of humans.The developed assay indicates a concept proof of a rapid, cost effective high throughput diagnostic tool for identification of major bacteria causing mastitis.

Validation of Ferret Luminex Cytokine Assays with serum from ferrets infected with flu, stimulated whole blood and PBMCs

Abstract Ferrets represent excellent models of infectious disease due to their natural susceptibility to many human respiratory viruses including influenza A virus (IAV), respiratory syncytial virus and severe acute respiratory syndrome (SARS). Ferrets can reproduce a similar course of disease and display similar symptoms of respiratory infection as humans, while mice do not exhibit key features of disease such as sneezing, nasal discharge and fever. There is pressing need to develop reagents to better understand and characterize the immune response to infection and support biomarker and countermeasure development through immunologic analyses of respiratory infections in the ferret. IL-1β, IL-2, IL-5, IL-6, IL-12 p40, IL-12p35, IL-17, IFNγ, TNFα, & IP-10 were expressed in HEK293 cells and purified. Monoclonal antibodies were generated and screened by Luminex for matched pairs to the recombinant protein and presumed positive samples. Capture antibodies were coupled to Luminex beads and detection antibodies were biotinylated. Serum from ferrets infected with flu had 10-fold higher levels of IP-10 and 3-fold higher levels of IL-6 than control serum. Stimulation of ferret whole blood with LPS showed increases in IL-2 and IL-6 at 2, 4 and 24 hrs. IL-12p40 and TNFα increased at 24hrs after stimulation with LPS. A validated cytokine panel in the study of immune related diseases in the ferret model. Supported by SBIR contract 75N93020C00022: Development of Ferret Reagents for use in the Characterization of Immune Responses to Respiratory Infections in the Ferret Model

Multiplex Glycan Bead Array (MGBA ) for High Throughput and High Content Analyses of Glycan-Binding Proteins Including Natural Anti-Glycan Antibodies.

We present here detailed protocols for the newly developed multiplex glycan bead array (MGBA) for the high throughput and high content analyses of various glycan-binding proteins including anti-glycan antibodies. This platform takes advantage of the commercially available Luminex beads to construct glycan arrays that are easily customizable at will and anytime by researchers. The platform allows the simultaneous analyses of up to 500 glycans and 384 samples at a time. By using multiple arrays, a researcher can analyze thousands of glycans and tens of thousands of samples within a short period. The assay is highly sensitive, specific, reproducible, economic, and fast. Furthermore, the bead array platform is approved for use in clinical settings, speeding up the translation of laboratory discoveries into patient care.

High-Throughput Strategies for the Discovery of Anticancer Drugs by Targeting Transcriptional Reprogramming.

Transcriptional reprogramming contributes to the progression and recurrence of cancer. However, the poorly elucidated mechanisms of transcriptional reprogramming in tumors make the development of effective drugs difficult, and gene expression signature is helpful for connecting genetic information and pharmacologic treatment. So far, there are two gene-expression signature-based high-throughput drug discovery approaches: L1000, which measures the mRNA transcript abundance of 978 “landmark” genes, and high-throughput sequencing-based high-throughput screening (HTS2); they are suitable for anticancer drug discovery by targeting transcriptional reprogramming. L1000 uses ligation-mediated amplification and hybridization to Luminex beads and highlights gene expression changes by detecting bead colors and fluorescence intensity of phycoerythrin signal. HTS2 takes advantage of RNA-mediated oligonucleotide annealing, selection, and ligation, high throughput sequencing, to quantify gene expression changes by directly measuring gene sequences. This article summarizes technological principles and applications of L1000 and HTS2, and discusses their advantages and limitations in anticancer drug discovery.

Multiplex detection of antibodies to Chikungunya, O'nyong-nyong, Zika, Dengue, West Nile and Usutu viruses in diverse non-human primate species from Cameroon and the Democratic Republic of Congo.

BackgroundEpidemic arbovirus transmission occurs among humans by mosquito bites and the sylvatic transmission cycles involving non-human primates (NHPs) still exists. However, limited data are available on the extent in NHPs infections and their role. In this study, we have developed and validated a high-throughput serological screening tool to study the circulation of multiple arboviruses that represent a significant threat to human health, in NHPs in Central Africa.Methodology/Principal findingsRecombinant proteins NS1, envelope domain-3 (DIII) for the dengue (DENV), yellow fever (YFV), usutu (USUV), west nile (WNV) and zika (ZIKV) and envelope 2 for the chikungunya (CHIKV) and o'nyong-nyong (ONNV) were coupled to Luminex beads to detect IgG directed against these viruses. Evaluation of test performance was made using 161 human sera of known arboviral status (66 negative and 95 positive). The sensitivity and specificity of each antigen were determined by statistical methods and ROC curves (except for ONNV and USUV). All NS1 antigens (except NS1-YFV), CHIKV-E2 and WNV-DIII had sensitivities and specificities > 95%. For the other DIII antigens, the sensitivity was low, limiting the interest of their use for seroprevalence studies. Few simultaneous reactions were observed between the CHIKV+ samples and the NS1 antigens to the non-CHIKV arboviruses. On the other hand, the DENV+ samples crossed-reacted with NS1 of all the DENV serotypes (1 to 4), as well as with ZIKV, USUV and to a lesser extent with YFV. A total of 3,518 samples of 29 species of NHPs from Cameroon and the Democratic Republic of Congo (DRC) were tested against NS1 (except YFV), E2 (CHIKV/ONNV) and DIII (WNV) antigens. In monkeys (n = 2,100), the global prevalence varied between 2 and 5% for the ten antigens tested. When we stratified by monkey’s biotope, the arboreal species showed the highest reactivity. In monkeys from Cameroon, the highest IgG prevalence were observed against ONNV-E2 and DENV2-NS1 with 3.95% and 3.40% respectively and in DRC, ONNV-E2 (6.63%) and WNV-NS1 (4.42%). Overall prevalence was low in apes (n = 1,418): ranging from 0% for USUV-NS1 to 2.6% for CHIKV-E2. However, a very large disparity was observed among collection site and ape species, e.g. 18% (9/40) and 8.2% (4/49) of gorillas were reactive with CHIKV-E2 or WNV-NS1, respectively in two different sites in Cameroon.Conclusions/SignificanceWe have developed a serological assay based on Luminex technology, with high specificity and sensitivity for simultaneous detection of antibodies to 10 antigens from 6 different arboviruses. This is the first study that evaluated on a large scale the presence of antibodies to arboviruses in NHPs to evaluate their role in sylvatic cycles. The overall low prevalence (<5%) in more than 3,500 NHPs samples from Cameroon and the DRC does not allow us to affirm that NHP are reservoirs, but rather, intermediate hosts of these viruses.

Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays

BackgroundFew bead-based multiplex assays have been described that detect antibodies against the protozoan parasite Toxoplasma gondii in large-scale seroepidemiological surveys. Moreover, each multiplex assay has specific variations or limitations, such as the use of truncated or fusion proteins as antigens, potentially masking important epitopes. Consequently, such an assay must be developed by interested groups as none is commercially available.ResultsWe report the bacterial expression and use of N-terminal fusion-free, soluble, in vivo biotinylated recombinant surface antigens SAG1 and SAG2A for the detection of anti-T. gondii IgG antibodies. The expression system relies on three compatible plasmids. An expression construct produces a fusion of maltose-binding protein with SAG1 (or SAG2A), separated by a TEV protease cleavage site, followed by a peptide sequence recognized by E. coli biotin ligase BirA (AviTag), and a terminal six histidine tag for affinity purification. TEV protease and BirA are encoded on a second plasmid, and their expression leads to proteolytic cleavage of the fusion protein and a single biotinylated lysine within the AviTag by BirA. Correct folding of the parasite proteins is dependent on proper disulfide bonding, which is facilitated by a sulfhydryl oxidase and a protein disulfide isomerase, encoded on the third plasmid. The C-terminal biotinylation allowed the oriented, reproducible coupling of the purified surface antigens to magnetic Luminex beads, requiring only minute amounts of protein per determination. We showed that an N-terminal fusion partner such as maltose-binding protein negatively influenced antibody binding, confirming that access to SAG1’s N-terminal epitopes is important for antibody recognition. We validated our bead-based multiplex assay with human sera previously tested with commercial diagnostic assays and found concordance of 98–100% regarding both, sensitivity and specificity, even when only biotinylated SAG1 was used as antigen.ConclusionsOur recombinant in vivo-biotinylated T. gondii antigens offer distinct advantages compared to previously described proteins used in multiplex serological assays for T. gondii. They offer a cheap, specific and sensitive alternative to either parasite lysates or eukaryotic-cell expressed SAG1/SAG2A for BBMA and other formats. The described general expression strategy can also be used for other antigens where oriented immobilization is key for sensitive recognition by antibodies and ligands.

Fluorescent bead-based serological detection of Toxoplasma gondii infection in chickens

BackgroundFree-ranging chickens are often infected with Toxoplasma gondii and seroconvert upon infection. This indicates environmental contamination with T. gondii.MethodsHere, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests, PCRs and bioassay in mice.ResultsIn experimentally infected chickens, the vast majority (98.5%, n = 65/66) of birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n = 45/45). Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISASL, n = 61/65; or TgSAG1-ELISASH, n = 60/65), or positive in an immunofluorescence assay (IFAT, n = 64/65) and in a modified agglutination test (MAT, n = 61/65). All non-inoculated control animals (n = 28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% confidence interval, CI: 90.7–99.9%) and specificity (100%; 95% CI: 85.0–100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%; 95% CI: 77.1–98.9%), while all bioassay- or PCR-negative chickens remained negative (100%; 95% CI: 85.0–100%).ConclusionsThe TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent a component in future multiplex assays for broad serological monitoring of poultry and other farm animals for various pathogens.

Better ABO Antibody Detection Tools to Facilitate ABO-Incompatible Transplant Risk Assessment

Accurate characterization of ABO-Ab is critical to assess impact in ABOi transplantation. The current ABO-Ab detection method using erythrocyte agglutination is limited by lack of ABO-subtype specificity, imprecise ABO-Ab isotype differentiation, and poor reproducibility. We previously developed an ABO-glycan microarray to address these limitations. Our aim here was to create a solid phase bead assay for ABO-Ab analysis. ABO A- and B-subtype antigens (I,II,III,IV,V,VI) were coupled to Luminex beads and quantified using monoclonal ABO-Ab. Bovine serum albumin was coupled as a negative control bead. IgG and IgM isotypes with specificities for ABO A-subtypes were measured and compared in healthy adult plasma (n=28) by mean fluorescence intensity (MFI). These samples were also tested on the glycan array and by red cell agglutination. ABO-A and B-subtype-specific antibodies were detected, with high degree of variability in MFI values between subjects. IgG and IgM ABO-A and B-Ab were detectable in all non-AB subjects, although MFIs were low in some cases by both bead and array (Figure 1A). Group A and B subjects had similar IgM ABO-A-Ab levels to group O subjects, but lower IgG levels. The quantity of IgM isotype antibodies did not predict IgG level (Figure 1B). IgM Ab alone did not predict the RBC agglutination titre, as some subjects had predominantly IgG ABO-Ab. Initial results of our Luminex ABO-Ab assay are promising for clinical laboratory use, where bead-based assays are already used for HLA-Ab. The specificity of this assay will allow precise assessment of ABO-Ab to antigen subtypes, known to be expressed differently in cardiac endothelium than erythrocytes (1). The ability to measure IgM and IgG ABO-Ab makes it possible to evaluate of the role of each in potential graft damage; isotype differentiation may be particularly relevant in the setting of plasmapheresis, which more efficiently removes IgM than IgG antibodies. 1. Jeyakanthan, M et al. AJT 2016; 16: 1548-1558.

Graft Immunocomplex Capture Fluorescence Analysis Can Detect Intragraft Anti–Major Histocompatibility Complex Antibodies in Mice Cardiac Transplant

BackgroundImmunocomplex capture fluorescence analysis has recently been applied as a method for detection of intragraft donor-specific anti–major histocompatibility complex (MHC) antibodies (DSA) in humans. Although intragraft DSA in humans is an intense topic of investigation, there is no report to assess intragraft DSA in murine organ transplantation. MethodsA model of presensitized mouse cardiac transplantation by donor splenocytes was used. To capture mouse MHC, anti-MHC class I/II antibodies were immobilized on Luminex beads. The MHC/DSA complexes were captured by the Luminex beads followed by detection of phycoerythrin-conjugated antimouse IgG antibodies where DSA had already reacted with the allograft in vivo. ResultsLuminex beads were capable of detecting class I DSA in the cardiac allograft, though results for class II DSA were negative. Immunohistochemical investigation revealed that cardiac allografts had abundant MHC class I expression but only minor expression of MHC class II. Furthermore, MHC/class II DSA complexes were successfully detected in splenocytes and serum from a presensitized recipient. ConclusionsThese data suggested that graft immunocomplex capture fluorescence analysis can be also applied in murine cardiac transplantation. This novel application in mice would accelerate our comprehension of DSA through mechanistic studies.

Interaction between maternally derived antibodies and heterogeneity in exposure combined to determine time-to-first Plasmodium falciparum infection in Kenyan infants

BackgroundStudies of the association between the level of anti-malarial antibody and protection from malaria infection can yield conflicting results if they fail to take into account differences in the malaria transmission rate. This can occur because high malaria exposure may drive high antibody responses, leading to an apparent positive association between immune response and infection rate. The neonatal period provides a unique window to study the protective effects of antibodies, because waning maternally-derived antibodies lead to different levels of protection with time.MethodsThis study uses data from two well-defined infant cohorts in Western Kenya with different burdens of malaria transmission. Survival models were used to assess how the magnitude of maternally derived malaria-specific IgG antibody (to 24 malaria antigens measured using Luminex beads) affected the time-to-first Plasmodium falciparum infection (detected by PCR). In addition, mathematical models were used to assess how the frequency of malaria infection varied between the cohorts with different exposure levels.ResultsDespite differences in underlying malaria incidence in the two regions, there was no difference in time-to-first malaria infection between the cohorts. However, there was a significant period of protection observed in children with high initial MSP1 (42 kDa fragment)-specific antibody levels, but this protection was not observed in children with low antibody levels. Children from the high transmission cohort had both longer initial periods of protection from malaria (attributable to higher initial antibody levels), but more rapid time-to-first-infection once malaria specific maternal antibodies declined below protective levels (attributable to higher exposure rates).ConclusionThis study demonstrates the complex interaction between passive (maternally-derived) immunity and the degree of malaria exposure in infants. Children from regions of high malaria transmission had higher levels of maternally-derived antibodies in early life, which led to a significant protection for several months. However, once this immunity waned, the underlying higher frequency of infection was revealed. A better understanding of the interaction between malaria exposure, immunity, and transmission risk will assist in identifying protective immune responses in P. falciparum infection.

Development and evaluation of a BK polyomavirus serotyping assay using Luminex technology

BackgroundThe BK polyomavirus (BKPyV) is subdivided into four genotypes. The consequences of each genotype and of donor-recipient genotype (mis)match for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant recipients (KTRs) are unknown. ObjectivesTo develop and evaluate a genotype-specific IgG antibody-based BKPyV serotyping assay, in order to classify kidney transplant donors and recipients accordingly. Study designVP1 antigens of six BKPyV variants (Ib1, Ib2, Ic, II, III and IV) were expressed as recombinant glutathione-s-transferase-fusion proteins and coupled to fluorescent Luminex beads. Sera from 87 healthy blood donors and 39 KTRs were used to analyze seroreactivity and serospecificity against the different BKPyV genotypes. Six sera with marked BKPyV serotype profiles were analyzed further for genotype-specific BKPyV pseudovirus neutralizing capacity. ResultsSeroreactivity was observed against all genotypes, with seropositivity rates above 77% comparable for KTRs and blood donors. Strong cross-reactivity (r > 0.8) was observed among genotype I subtypes, and among genotypes II, III and IV. Seroresponses against genotypes I and IV seemed genuine, while those against II and III could be out(cross)competed. GMT (Luminex) and IC50 (neutralization assay) values showed good agreement in determining the genotype with the strongest seroresponse within an individual. ConclusionsDespite some degree of cross-reactivity, this serotyping assay seems a useful tool to identify the main infecting BKPyV genotype within a given individual. This information, which cannot be obtained otherwise from nonviremic/nonviruric individuals, could provide valuable information regarding the prevalent BKPyV genotype in kidney donors and recipients and warrants further study.

OR34 Modernizing ABO antibody detection: Development of a bead-based ABO antibody detection assay

Aim Accurate, clinically relevant characterization of ABO antibodies (ABO-Ab) is critical to assess their impact in ABO incompatible transplantation. The current ABO-Ab detection method using erythrocyte agglutination is limited by lack of ABO subtype specificity, difficulty in ABO-Ab isotype differentiation, and poor reproducibility. We previously developed a slide micro-array method for ABO-Ab analysis to address these limitations. Our aim was to create a similar bead solid phase assay. Methods ABO A subtype antigens (I, II, III, IV, V, VI) were coupled to Luminex beads and quantified using monoclonal ABO-Ab. Bovine serum albumin and alpha-Gal antigen were coupled as negative/positive control beads, respectively. Optimal plasma and anti-human IgG and IgM labelling-antibody dilutions were determined. IgG and IgM isotypes with specificities for ABO A-subtypes were measured (n = 40 healthy donors) by mean fluorescent intensity (MFI). Levels of IgG vsIgM ABO-A-Ab were compared (Wilcoxon signed-rank test). Preliminary positive MFI thresholds for each ABO A-subtype bead were calculated based on ABO-A-Ab in ABO A control plasma. Results Variation in ABO-Ab levels between ABO A-subtypes was detected. There was no significant difference between IgG and IgM ABO-Ab levels in paired MFI for all ABO types. IgG and IgM ABO-Ab are clearly detectable in all non-ABO A controls. Calculated MFI thresholds accurately interpreted positive ABO-A-Ab in ABO O samples for the IgG assay and most beads in the IgM assay. ABO B controls had similar IgM ABO-A-Ab levels to ABO O controls but lower levels of IgG. Reactivity to alpha-Gal was present but inconsistent. Conclusions This bead-based method successfully measures ABO-A-Ab and shows promise for clinical laboratory implementation. The specificity of these solid-phase assays will facilitate accurate assessment of ABO-Ab to subtypes, which are known to be expressed differently in endothelium than on erythrocytes. The ability to accurately measure both IgM and IgG ABO antibodies makes it possible to evaluate of the role of each ABO-Ab isotype in transplantation; this may be particularly relevant in the setting of plasmapheresis, which more efficiently removes IgM antibodies. Further optimization of this assay, controls, and ABO B and ABO H panel development are underway.

OR45 Proof of principle: Bead-based ABO antibody assessment

Aim ABO antibody (ABO-Ab) titration is performed for ABO-incompatible (ABOi) transplantation management and is traditionally assessed by hemagglutination. However, hemagglutination cannot readily differentiate Ab isotype, nor distinguish between Abs to ABO-subtype antigens, which are expressed differently on organs vs erythrocytes. Our goal was to explore Luminex as a potential platform for the detection of IgG and IgM antibodies to ABO subtype antigens. Methods Phase I: Human blood group antigen subtype A-II was synthesized and conjugated to bovine serum albumin (BSA). BSA-A-II conjugates were coupled to Luminex beads. Coupling was assessed using phycoerythrin (PE)-labeled mouse monoclonal IgG and IgM anti-human ABO-A. Increasing BSA-A-II antigen concentrations were tested to optimize MFI. A Luminex-200 was used for bead acquisition. Phase II: Antigens ABO A-III and A-IV were similarly tested (alone and multiplexed) using additional Luminex beads. Phase III: Dilutions of PE-labeled anti-human IgG conjugate were tested against dilutions of anti-ABO-A plasma (blood group B donor) in a checkerboard titration. BSA-A-II coupled beads were used. Results Bead coupling was optimal at 5ug of BSA-A-II antigen. The coupling of BSA-A-II, -III and -IV was successful as demonstrated by a linear increase in MFI with increasing concentration of primary antibody (Figure). Beads were successfully labeled with monoclonal ABO-Ab when multiplexed. The assay appears most sensitive with the lowest background when patient plasma is diluted 1/25. Conclusions HLA laboratories are uniquely situated to assess ABO antibodies by Luminex as expertise and instrumentation already exist. This novel assay has the potential to be adopted by clinical HLA laboratories for rapid, specific, and sensitive assessment of IgM/IgG ABO subtype-specific ABO antibodies in ABOi transplantation. We continue to optimize the assay, include additional controls, and assess additional ABO subtypes. We are also testing flow-based bead platforms. Download : Download high-res image (185KB) Download : Download full-size image

P022 Variations in surface expression and stability among human leukocyte antigens (HLA-B)

Aim Among HLA class I (HLA-I) molecules, HLA-B allotypes are the most polymorphic and have dominant influences upon disease outcomes. HLA-B allotypes are known to differ markedly in their intracellular folding and peptidome characteristics. The Aim of this study was to assess whether intracellular assembly or peptidome variations influence the cell surface expression levels or stabilities of HLA-B allotypes. Methods Commercial HLA-Bw4 and HLA-Bw6 antibodies were assessed for HLA-I specificities using Luminex beads, and antibody binding differences were quantified. 244 healthy donors were recruited and HLA genotyping was performed using next generation sequencing (NGS). Donors were selected for further measurements based on heterozygosity for the Bw4 and Bw6 epitopes within their HLA-B antigens, and based on the absence of cross-reactive HLA-A antigens. Using quantitative flow cytometry, antibody binding capacity (ABC) values were measured on freshly isolated lymphocyte subsets using HLA-Bw4, HLA-Bw6 and total class I-specific antibodies. For each selected donor, HLA-I ABC measurements were undertaken at multiple time points. For HLA-B alleles with measurements from at least three donors, averaged ABC values for HLA-Bw4 and HLA-Bw6 were analyzed directly or as a ratio relative to total HLA-I, after taking into account relevant antibody recognition differences. For some HLA-Bw4 and HLA-Bw6 allotypes, cell surface stabilities were also assessed on freshly isolated lymphocytes in the presence of brefeldin A. Results Statistically significant allele-dependent differences are measured in cell surface HLA-B expression levels. For some HLA-B allotypes, lower expression could be related to reduced cell surface stability. Cell surface expression and stability differences appear to be related to the individual peptide-binding characteristics of HLA-B, rather than diversities (promiscuities) of their peptide repertoires. Conclusions Overall, these studies indicate allele-dependent variations in cell surface expression and stability of HLA-B, which in some cases correlate with previously known disease progression outcomes in human immunodeficiency virus (HIV) infections.

OR49 Effect of everolimus immunotherapy on HLA-antibody mediated activation of endothelial cells in heart transplantation

Aim Donor-specific HLA antibodies (DSA) have been implicated in the process of acute and chronic antibody-mediated rejection (AMR) through their capacity to activate and injure the capillary endothelium of vascularized allografts. Ligation of HLA molecules on endothelial cells (EC) with HLA alloantibodies stimulates phosphorylation of S6 ribosomal protein (pS6RP) via the mechanistic target of rapamycin (mTOR). We hypothesize that expression of pS6RP in the capillary endothelium of allografts is a useful biomarker of rejection and can be used to assess the clinical efficacy of the mTOR inhibitor everolimus. Methods 188 heart transplant patients from Novartis sponsored 2310 clinical multicenter trial were included in this study. Among them, 118 recipients were treated with reduced cyclosporine (CsA), everolimus (1.5 or 3.0 mg/day) and steroids; 70 were treated with mycophenolate mofetil (MMF, 3 g/day), standard CsA and steroids. AMR and acute cellular rejection (ACR) were diagnosed according to ISHLT criteria. Capillary endothelial staining of pS6RP was scored as 0–4 on endomycardial biopsies by immunohistochemistry. DSA was measured using single antigen luminex beads. Result 44.3% patients in MMF group and 37.6% patients in everolimus group developed DSA, predominantly against HLA class II (p = 0.29, Fisher’s exact test). DSA producers demonstrated significantly higher pS6RP score (1.04 ± 0.07 vs 0.73 ± 0.05, p ⩾ 2R and pAMR, 39.5% vs 25.2%, p = 0.05) compared to patients without DSA. Everolimus treatment attenuated pS6RP (everolimus: 0.65 ± 0.06 vs MMF: 0.97 ± 0.07, p Conclusions Everolimus therapy reduces HLA DSA-induced EC activation/injury in heart transplants via blocking mTOR signaling and lowers the incidence of rejection and transplant vasculopathy.

Low prevalence of human papillomavirus in head and neck squamous cell carcinoma in the northwest region of the Philippines.

BackgroundGeographic heterogeneity of human papillomavirus (HPV) involvement in head and neck squamous cell carcinoma (HNSCC) has been observed over the last few years. This trend has not been evaluated in the Philippines. Hence, this study aims to provide for the first time a data on the prevalence of HPV in HNSCC in the northwestern region of the Philippines.MethodsTwo hundred one (201) biopsy samples (179 formalin fixed paraffin embedded and 22 fresh frozen) from 163 Filipino HNSCC cases (oral cavity = 88; larynx = 60; oropharynx = 15) diagnosed between 2003 to 2013 were initially included in this study. HPV DNA was detected by two methods: (1) BSGP5+/6+-PCR/ multiplex human papillomavirus genotyping and (2) TaqMan probes-based real-time qPCR. Presence of HPV type-specific transcripts were also analyzed by reverse transcription-PCR with subsequent hybridization to oligonucleotide probes coupled to Luminex beads. Co-amplification of the β-globin and ubiquitin C genes served as internal positive controls for DNA and RNA analyses, respectively.Results and conclusionsOf the 163, 82 (50.3%) cases had at least one tissue sample that was valid for molecular analysis. Only two of the DNA valid cases (2.4%) were HPV DNA-positive (HPV11 and HPV33). All HPV mRNA assays rendered negative results except for HPV11 transcripts. Results of this study may indicate that there is probably very low prevalence of HPV-associated HNSCC among Filipino adults living in a rural region of the Philippines. This study could serve as a benchmark for designing follow-up studies that would assess possible changes in trends of HNSCC among Filipinos in different ethnic regions of the country, especially urban areas in which the population is expected to adapt Western style sexual behavior. A prospective sampling of fresh frozen tissue is also highly recommended to ensure better molecular analyses.