Luminex Beads Research Articles

Application of recombinant GPⅢa combined Luminex beads for the detection of HPA-1a antibody

To generate recombinant GPⅢa as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody. The full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer. DNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GPⅢa was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL). The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible. Recombinant GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.

Multiplex Immunoassay Profiling of Serum in Psychiatric Disorders.

Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labour than in single immunoassays. This chapter details the methods to develop and manufacture a 5-plex immunoassay for the Luminex® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. An example will be given for the analysis of five hormones (glucagon-like peptide 1, growth hormone, insulin, leptin and thyroid-stimulating hormone) in serum samples from schizophrenia patients and controls.

Multiplex Immunoassay Profiling.

Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labor than single immunoassays. This chapter details the methods to develop and manufacture multiplex assays for the Luminex® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. The assay optimization, detection of cross-reactivity, and minimizing antibody interactions and matrix interferences will be addressed.

A Molecular Analysis of the Shared Epitope Hypothesis: Binding of Arthritogenic Peptides to DRB1*04 Alleles.

The shared epitope hypothesis posits that amino acids QR/KRAA in positions 70-74 of the DRΒ1 chain are responsible for rheumatoid arthritis susceptibility. However, even DRB1*04 alleles containing the shared epitope vary greatly with respect to degrees of susceptibility. This study was undertaken to conduct a molecular examination of the shared epitope hypothesis by measuring binding of arthritogenic peptides to susceptibility and resistance alleles. We measured binding of native and citrullinated forms of vimentin(66-78) and α-enolase(11-25) and noncitrullinated type II collagen(258-272) to 88 class II alleles on Luminex beads (which includes alleles of many varying degrees of susceptibility and resistance). We expressed DRΒ1*04:01, *04:02, and *08:01 in T2 cells and mutated DRΒ1*04:01 at positions 67, 70, 71, 74, and 86 to corresponding residues in DRB1*04:02, *04:03, *04:04, *04:05, and *08:01. Finally, we measured responses of 4 DRΒ1*04:01 restricted collagen(258-272) T cell hybridomas against wild-type DRΒ1*04:01, *04:02, and all mutated alleles. The most susceptible allele, DRΒ1*04:01, preferentially bound citrullinated vimentin(66-78) and citrullinated α-enolase(11-25) over the native forms. DRΒ1*04:02 exhibited no preference for citrullinated peptides, and *08:01 preferred native peptides. Similarly, DRB1*04:01 bound collagen(258-272) , but *04:02 and *08:01 did not. Mutating DRΒ1*04:01 at positions 70, 71, 74, and 86 to the corresponding residues in DRΒ1*04:02 or *08:01 dramatically reduced the specificity for citrullinated peptides and collagen(258-272) binding. These observations demonstrate that while amino acids at positions 70, 71, and 74 within the shared epitope in DRΒ1 mediate binding and T cell responses of arthritogenic peptides, position 86 outside the shared epitope also plays a critical role.

STAT3 inhibition prevents lung inflammation, remodeling, and accumulation of Th2 and Th17 cells in a murine asthma model

STAT3 drives development of Th17 cells and cytokine production by Th2 and Th17 cells, which contribute to asthma. Alternative asthma treatments are needed, especially for the Th17 phenotype. We sought to determine whether C188-9, a small-molecule STAT3 inhibitor, can block Th2 and Th17 cell expansion and cytokine production to prevent house dust mite (HDM)-induced airway inflammation and remodeling. Three groups of C57BL/6 mice were treated intranasally (IN) and intraperitoneally (IP) daily for 3 weeks with the following: (i) vehicle 1 IN and vehicle 2 IP, (ii) HDM IN and vehicle 2 IP, or (iii) HDM IN and C188-9 IP. Sections of lung were stained with Alcian Blue/PAS and examined microscopically. Total (t) STAT3, STAT3 phosphorylated on Y705 (pSTAT3), IL-17, IL-13, IL-5, and IL-4 levels were measured in lung protein extracts and serum using Luminex beads. Frequencies of Th2-type and Th17-type lymphocytes were assessed in lungs and bronchoalveolar lavage fluid (BALF) by multiparametric flow cytometry. HDM inhalation markedly increased airway goblet cell numbers and thickness of the epithelium and subepithelial smooth muscle layer, which was accompanied in the whole lung by increased pSTAT3, IL-4, IL-5, IL-13, and IL-17, and % CD4+ T cells that produce IL-5, IL-13, and IL-17. HDM inhalation also increased serum IL-4 and IL-17 levels and increased BALF % CD4+ T cells that produce IL-5 and IL-13. Remarkably, treatment with C188-9 normalized each endpoint. HDM-induced airway inflammation, remodeling, and Th2/Th17-type cell accumulation involve STAT3 activation that can be prevented by C188-9 treatment.

Analysis of Human Platelet Antigen-1 System Alloantibodies Using Recombinant GPIIIa Fragments Coupled to Luminex Beads

To detect platelet anti-HPA-1a and -1b antibodies using recombinant GPIIIa fragments coupled to Luminex beads. The sensitivity of 2 techniques, monoclonal antibody specific immobilization of platelet antigen (MAIPA) and Luminex bead assay, was compared using 12 twofold-serial dilutions (from neat to 1 in 2048) of an anti-HPA-1a WHO international standard. The specificity of Luminex assay to identify anti-HPA-1a and -1b antibodies was assessed using 8 negative or positive controls and 36 blinded samples provided by WHO Platelet Workshop. The sensitivity of MAIPA and Luminex bead assay to detect anti-HPA-1a was dilution 1/64 (i.e. 1.56 IU/ml) and far more than dilution 1/2048 (i.e. 0.049 IU/mL), respectively. The Luminex bead assay could specifically identify negative and positive controls of anti-HPA-1a and -1b. All results of 36 blinded samples by Luminex assay were accordant to reference results except one sample which contained high concentration antithetical antibody and resulted in false positive of anti-HPA-1b. Cross-reactivity was also not observed with the samples containing HLA, ABO or other platelet antibodies. The Luminex beads coupled with recombinant GPIIIa fragments can be used to detect HPA-1 system antibodies with sufficient sensitivity and specificity, that is suitable for the detection of platelet alloantibodies in clinical alloimmune thrombocytopenia.

Sensitive Multiplexed Quantitative Analysis of Autoantibodies to Cancer Antigens with Chemically S-Cationized Full-Length and Water-Soluble Denatured Proteins.

Humoral immune responses against tumor-associated antigens (TAAs) or cancer/testis antigens (CTAs) aberrantly expressed in tumor cells are frequently observed in cancer patients. Recent clinical studies have elucidated that anticancer immune responses with increased levels of anti-TAA/CTA antibodies improve cancer survival rates. Thus, these antibody levels are promising biomarkers for diagnosing the efficiency of cancer immunotherapy. Full-length antigens are favored for detecting anti-TAA/CTA antibodies because candidate antigen proteins contain multiple epitopes throughout their structures. In this study, we developed a methodology to prepare purified water-soluble and full-length antigens by using cysteine sulfhydryl group cationization (S-cationization) chemistry. S-Cationized antigens can be prepared from bacterial inclusion bodies, and they exhibit improved protein solubility but preserved antigenicity. Anti-TAA/CTA antibodies detected in cancer patients appeared to recognize linear epitopes, as well as conformational epitopes, and because the frequency of cysteine side-residues on the epitope-paratope interface was low, any adverse effects of S-cationization were virtually negligible for antibody binding. Furthermore, S-cationized antigen-immobilized Luminex beads could be successfully used in highly sensitive quantitative-multiplexed assays. Indeed, patients with a more broadly induced serum anti-TAA/CTA antibody level showed improved progression-free survival after immunotherapy. The comprehensive anti-TAA/CTA assay system, which uses S-cationized full-length and water-soluble recombinant antigens, may be a useful diagnostic tool for assessing the efficiency of cancer immunotherapy.

OR43 Four DRβ1 amino acids mediate preferential binding of citrullinated peptides and collagen

Aim Several polymorphic amino acids within the peptide-binding groove of the DR β 1 molecule correlate with rheumatoid arthritis disease susceptibility and resistance. We have previously demonstrated through assessment of peptide binding to 97 HLA class II alleles on Luminex beads that susceptible alleles exhibit a preference for citrullinated peptides over native. In this study, we assess the importance of these amino acids in binding and T cell responses to potentially arthritogenic peptides to alleles containing mutations in the peptide binding groove. Methods We measured binding of native and citrullinated forms of vimentin66–78 and α-enolase11–25 and non-citrullinated type II collagen259–273, to DRβ1*04:01, DRβ1*04:02 and DRβ1*08:01 wild type alleles and DRβ1*04:01 with the following mutations: L67I, L67F, Q70D, K71E, K71R, A74L and G86V. We also measured T cell hybridoma responses to type II collagen259–273 when presented on the aforementioned alleles. Results The most susceptible allele, DRβ1*04:01, preferred citrullinated vimentin66–78 and citrullinated α-enolase11–25 over the native forms. Resistant alleles DRβ1*04:02 and *08:01 exhibited either no preference or preferred the native forms of these peptides. Similarly, collagen259–273 bound to DRβ1*04:01, but not to *04:02 or *08:01. Individually mutating susceptible DRβ1*04:01 at positions 70, 71, 74 and 86 to the corresponding resistance residues in DRβ1*04:02 or *08:01 dramatically reduced the specificity for citrullinated peptides and collagen. T cell hybridomas responded to collagen259–273 presented on DRβ1*04:01 and DRβ1*04:01 with mutations at position 67. However, despite binding being conserved among some mutations, negatively charged amino acids at positions 70 and 71 eliminated the T cell response to collagen. Conclusions These observations suggest that Q70, K71, A74 and G86 in DRβ1 mediate binding preference of both citrullinated and non-citrullinated arthritogenic peptides and are important for T cell responses. Download : Download full-size image

Abstract LB-261: Mechanism of action of MCLA-128, a humanized bispecific IgG1 antibody targeting the HER2:HER3 heterodimer

Abstract Background: MCLA-128 is an ADCC-enhanced humanized common light chain bispecific IgG1 antibody that targets the HER2:HER3 dimer with nanomolar affinity, potently inhibiting tumor growth in vitro and in vivo. MCLA-128 shows superior activity to the combination trastuzumab/ pertuzumab and HER3 targeting monoclonal antibodies and is currently being evaluated in a Phase I clinical trial. This study investigated the mechanism of action of MCLA-128. Methods: Phosphorylation of HER receptors and downstream signaling molecules was studied in vitro and in vivo on HER2-amplified cancer cell lines by Pathscan arrays, luminex beads and Western blot analysis. Inhibition of MCLA-128 cell growth in combination with tyrosine kinase inhibitors and small molecules targeting the MAPK and PI3 kinase/Akt pathway was determined by proliferation inhibition and high content imaging assays. The potential effect of MCLA-128 on primary cardiomyocytes in the presence of Doxorubicin was analyzed by measuring ATP. Binding of MCLA-128 to a panel of cell lines in comparison to HER2 and HER3 antibodies was determined by FACS. Results: In contrast to other HER2 and HER3 targeted agents, only MCLA-128 inhibited phosphorylation of HER3 and downstream Akt and ERK in HER2 amplified cell lines cultured with high concentrations of heregulin in vitro. In xenograft studies, growth inhibition of the trastuzumab-resistant cell line JIMT-1 by MCLA-128 was correlated with reduced HER2:HER3 dimerization and a profound inhibition of the PI3K pathway. Synergistic growth inhibition in vitro was observed when tyrosine kinase inhibitors or inhibitors of the PI3K pathway were added to HER2 amplified cancer cells in the presence of MCLA-128. MCLA-128 did not show any evidence of cardiotoxicity in vitro in contrast to trastuzumab. MCLA-128 binds and coats breast cancer cell lines with differing levels of HER2 expression more efficiently in comparison to monospecific HER2 or HER3 monoclonal antibodies. Conclusions: The unique simultaneous targeting of MCLA-128 to HER2 and HER3 on HER2-overexpressing breast cancer cells leads to severe impairment of PI3K signaling and reduced cell growth whereas proliferation of primary cardiomyocytes is unaffected. The enhanced coating effect of MCLA-128 also supports its ADCC activity. Citation Format: Cecile Geuijen, Eric Rovers, Tristan Gallenne, David Maussang-Detaille, Arjen Kramer, Nellie Nieuwenhuizen, Carina Clements, Katinka van Zoest, Roy Nijhuis, Therese Visser, Renate Den Blanken-Smit, Willem Bartelink, Vanessa Zondag-van der Zande, Linda Kaldenberg, Pieter-Fokko van Loo, Rob Roovers, Robert Doornbos, Leo Price, Stefan Braam, Setareh van Driel, Lex Bakker, Ton Logtenberg, John de Kruif, Mark Throsby. Mechanism of action of MCLA-128, a humanized bispecific IgG1 antibody targeting the HER2:HER3 heterodimer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-261. doi:10.1158/1538-7445.AM2015-LB-261

LBP01: DIFFERENT STROKES FOR DIFFERENT FOLKS: CONCORDANCE AND DISCORDANCE IN ANTI-HLA ANTIBODY TESTING

Background Correct identification of the specificity of antibodies directed against HLA using Single Antigen Luminex Beads (SALB) is essential in current HLA laboratory practice for transplantation. The cumulative data collected by the Proficiency Testing (PT) Program from hundreds of laboratory submissions over years of activity provide an invaluable opportunity to study concordance in antibody testing in a broad group of HLA laboratories. The aim of this study was to investigate the magnitude of concordance and discordance among laboratories in testing for anti-HLA antibodies using SALB. Method 35 sera were distributed by the ASHI PT Program to HLA laboratories worldwide. An average of 124 laboratories reported results derived from SALB assays in each of the 7 surveys described (range: 116–132). Laboratories were free to choose their test methods and standard operating procedures. We analyzed 4335 test results submitted between April 2010 and April 2013 by participating laboratories. Results SALB was used by 94% of the participating laboratories, yet concordant assignment of antibody specificity was imperfect. For each serum, the assignment of an average of 10 antibody specificities was discordant. Disagreement was observed for antibodies directed against common as well as uncommon antigens. The assignment of an average of 15 antibody specificities in each “positive” serum appeared to be influenced by vendor-dependent causes. Inter-vendor concordance was lower than intra-vendor concordance, indicating that vendor dependent factors may be a central cause for disagreement. Conclusions Our study illustrates the prevalence of concordance and discordance, also affected by unpremeditated causes, in reporting SALB antibody results. We have shown that several antibody specificities, some of them for very common HLA antigens, are frequently in disagreement between laboratories which tested the same serum to the extent that disagreement may be more prevalent than agreement. Insufficient concordance and standardization in antibody testing may have practical implications for organ allocation and organ sharing programs.

LBP22: STRONG DONOR SPECIFIC ANTI-HLA DR53 ANTIBODY DETECTED BY SINGLE-ANTIGEN BEADS SHOULD NOT ALWAYS PREVENT TRANSPLANTATION

Background Donor-specific antibodies towards HLA-DR51/52/53 were reported both as risk factors for allograft rejection, and as specificities with poor response to desensitization therapy. Here we present a case with strong antibody towards HLA-DR53 who was successfully transplanted. Case study. 47-year-old male renal transplant candidate with cPRA of 68%, was repeatedly detected with anti-HLA DR53 antibodies by Luminex single-antigen beads. HLA-DR53 was a mismatched antigen for his potential living donor (Table 1). Antibody IgG strength for a-HLA DR53 ranged between 7500 and 10000 MFI over one-year period. At these MFI values, our lab usually expects a positive cytotoxic B-cell crossmatch, or at least a positive B-cell flow crossmatch. However, both cytotoxic and flow crossmatches with his living donor were negative. To address this issue, we tested the living donor with other two sera known for a-HLA DR 53 activity, both in neat, and 1:4 and 1:8 dilutions, by flow crossmatch. All these combinations provided positive results of > 180 MCS above cutoff (1024 scale). Alternatively, candidate recent and peak sera was flow crossmatched with other two HLA-DR 53 cells, giving again negative results. Under the hypothesis of cryptic epitope DR53 reactivity, we also tested candidate sera with Luminex beads of different antigenic source (mixed and PRA), which have shown no DR53 reactivity. Under these circumstances, we concluded that the virtual crossmatch between the transplant candidate and his living donor was negative. Post transplant monitoring shows no signs of rejection at one year of follow-up. Conclusion Although solid-phase methods for antibody detection became mandatory in renal transplantation, a combination of technologies, rather than a single one, would be more adequate for antibody detection and identification. Table 1 ABO A B C DR DRw DQ DP Patient B 1 80 71 72 2 7 11 8 52 7 0301 0101 Donor 1 B 2 23 27 51 1 2 11 4 52 53 7 8 0401

High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology

Mosquito-borne arboviruses are emerging world-wide as important human and animal pathogens. This makes assays for their accurate and rapid identification essential for public health, epidemiological, ecological studies. Over the past decade, many mono- and multiplexed assays targeting arboviruses nucleic acids have been reported. None has become established for the routine identification of multiple viruses in a “single tube” setting. With increasing multiplexing, the detection of viral RNAs is complicated by noise, false positives and negatives. In this study, an assay was developed that avoids these problems by combining two new kinds of nucleic acids emerging from the field of synthetic biology. The first is a “self-avoiding molecular recognition system” (SAMRS), which enables high levels of multiplexing. The second is an “artificially expanded genetic information system” (AEGIS), which enables clean PCR amplification in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon, improving their efficiency of hybridization on Luminex beads. When Luminex “liquid microarrays” are exploited for downstream detection, this combination supports single-tube PCR amplification assays that can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The assay differentiates between closely-related viruses, as dengue, West Nile, Japanese encephalitis, and the California serological group. The performance and the sensitivity of the assay were evaluated with dengue viruses and infected mosquitoes; as few as 6–10 dengue virions can be detected in a single mosquito.

Highly specific DNA detection employing ligation on suspension bead array readout

We show for the first time that monomerized rolling circle amplification (RCA) products can be directly detected with the Luminex suspension bead array readout without the need of PCR amplification. Furthermore, using monomerized RCA products to guide ligation of the detection oligonucleotide (DO) to barcode sequences on the magnetic Luminex beads, combined with efficient washing and increased measurement temperature, yields a higher signal to noise ratio. As a proof-of-principle, we demonstrate detection of pathogenic DNA sequences with high reproducibility, sensitivity and a dynamic range over four orders of magnitude. Using padlock probes in combination with bead suspension arrays opens up the possibility for highly multiplexed DNA targeting and readout.

Concordance and discordance in anti-HLA antibody testing

BackgroundCorrect identification of the specificity of antibodies directed against HLA using single antigen Luminex beads (SALB) is essential in current HLA laboratory practice for transplantation. The aim of this study was to investigate the magnitude of concordance and discordance among laboratories in testing for anti-HLA antibodies using SALB. Method35 sera were distributed by the ASHI Proficiency Testing Program to HLA laboratories worldwide. We analyzed 4335 test results submitted between April 2010 and April 2013 by participating laboratories. ResultsSALB was used by approximately 94% of the participating laboratories, yet concordant assignment of antibody specificity was imperfect. For each serum, the assignment of an average of 10 antibody specificities was discordant. Disagreement was observed for antibodies directed against common as well as uncommon antigens. The assignment of an average of 15 antibody specificities in each “positive” serum appeared to be influenced by vendor-dependent causes. Inter-vendor concordance was lower than intra-vendor concordance, indicating that vendor dependent factors may be a central cause for disagreement. ConclusionsOur study illustrates the prevalence of concordance and discordance, also affected by unpremeditated causes, in reporting SALB antibody results. Insufficient concordance and standardization in antibody testing may have practical implications for organ allocation and organ sharing programs.

Abstract 2869: A three-protein cytokine signature that correlates with the prognosis of osteosarcoma

Abstract Osteosarcoma (OS) is the most common malignant bone tumor in pediatrics. Despite the use of surgery and chemotherapy, the survival rate of patients who develop a metastatic disease or a poor response towards the chemotherapy remains dismal. Identification of novel biomarkers for early prognostication will facilitate risk-based stratification before the treatment is initiated, so that the high-risk patients can be treated more appropriately to increase their chance of survival. Others and we have previously reported that cytokines and chemokines were implicated in OS. In this study, we employed highly sensitive Luminex beads assays to investigate 90 cytokines/chemokines and receptors on a cohort of 40 OS plasma samples collected at diagnosis from the Texas Children's Hospital. The protein expression data were analysed to identify candidate biomarkers that correlate with the chemotherapy response, initial metastatic status, and overall survival. Then, the candidates were validated in a large set of 250 serum samples obtained from the Children's Oncology Group. Our results show that a three-protein signature consisting of CXCL9, CXCL10 and CXCL11, all ligands of the CXCR3 receptor, is significantly associated with survival. Interestingly, the CXCR3-axis has been previously implicated in an OS mouse model, but its role in human is still not known. Hence, the next step is to validate these prognostic biomarkers in an independent cohort of OS patient samples. In addition, we will test the expression of these molecules and their receptor in tumor samples to identify potential therapeutic targets. In summary, we characterized the expression of circulating cytokines/chemokines in osteosarcoma samples and identified candidate prognostic biomarkers that are linked to the CXCR3-axis. A further study of these molecules may allow us to develop a biomarker approach for early prognostication and a novel therapeutic approach for treating high-risk OS. Citation Format: Ricardo J. Flores, Yiting Li, Aaron Kelly, Ching Lau, Tsz-Kwon Man. A three-protein cytokine signature that correlates with the prognosis of osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2869. doi:10.1158/1538-7445.AM2014-2869

OR15: SUSCEPTIBLE HLA SHARED EPITOPES IN RHEUMATOID ARTHRITIS MEDIATE BINDING OF CITRULLINATED PEPTIDES TO THE MHC

Aim Rheumatoid Arthritis (RA) is a chronic inflammatory disorder affecting 1% of the world population and results in the eventual destruction of bone and joints. A two amino acid Shared Epitope (SE) LA67,74 in HLA-DRB1 is highly associated with RA and is also positively correlated with high levels of anti-citrullinated protein antibodies. In contrast, a single amino acid SE D70 in HLA-DRB1 is associated with resistance in a resistance dominance fashion. We have previously demonstrated through assessment of peptide binding to 91 HLA class II alleles on Luminex beads that citrullinated vimentin binds significantly over native to HLA-DRB1 alleles containing the LA67,74 epitope. In this study, we assess the importance of these HLA SE in binding of citrullinated peptides to alleles containing SE mutations. Methods We measured citrullinated and native vimentin binding by flow cytometry on T2 cells transduced with either DRB1∗04:01 Wild Type (WT), DRB1∗04:02 WT, or DRB1∗04:01 containing the single mutations L67I and Q70D in addition to DRB1∗04:01 containing both mutations. Results DRB1∗04:01 WT bound citrullinated vimentin significantly over native. DRB1∗04:02 bound both citrullinated and native vimentin equally. Mutagenesis of DRB1∗04:01 L67I resulted in a modest reduction of binding of citrullinated vimentin, but excluded native vimentin binding. A Q70D mutation however allowed both citrullinated and native vimentin binding. DRB1∗04:01 containing both epitope mutations showed binding comparable to DRB1∗04:02. Conclusions As observed in our Luminex peptide binding assay, we found that LA67,74 displays a preference for citrullinated peptides. In contrast, the D70 epitope confers no preference for citrullinated peptides. Our results suggest that the presence of LA67,74 in the HLA cannot accommodate arginine in the binding pocket, whereas D70 alleles allow both arginine and citrulline binding. This provides rationale for LA67,74 associated susceptibility to RA in addition to D70 associated resistance to RA.

P027 : DONOR SPECIFIC HLA ANTIBODIES DETECTED BY LUMINEX CANNOT PREDICT THE OUTCOME OF A CDC OR FLOW CYTOMETRIC CROSSMATCH IN AN INDIVIDUAL PATIENT

For clinical purposes it is necessary that before a renal transplantation takes place, the patient is screened for the presence of circulating HLA antibodies that may harm the transplanted kidney. Antibody analysis is nowadays often performed by testing the reactivity towards HLA molecules bound to Luminex beads. The prospective crossmatch, however, is performed either by the complement-dependent cytotoxicity assay (CDC) or by flow cytometric assays. In the current study we compared the outcome of the luminex single antigen (SA) analysis with respect to the presence of donor specific antibodies (DSA) and the crossmatch result of 125 patients. CDC crossmatch results (pos/neg) and flow cytometric T cell and B cell crossmatch results (mean channel shift) were related to anti-class I or anti-class II reactivity in luminex (MFI). Results On the population level, there was a significant association between the sum of the MFI’s of the donor reactive HLA class I and II antibodies and outcome of the CDC cross match. However, when the significance for individual patients was analysed, the situation is less clear. Numerous negative CDC crossmatches were observed in case of high DSA levels in luminex ( Fig. A ). With respect to the flow T cell crossmatch, a higher channel shift was observed when anti-donor luminex reactivity increased but, in general, the correlation was poor. Similar findings were observed for the flow B cell crossmatch ( Fig. B ). In conclusion, despite the presence of a clear correlation between DSA detected by Luminex and the crossmatch result on the population level, for an individual patient one cannot rely on measurement of DSA by luminex only. This observation is very important if one considers the introduction of a virtual crossmatch. A combination of techniques will be necessary in order to predict in a reliable way the outcome of a crossmatch in sensitized individuals.

Pre-transplant donor HLA-specific antibodies: Characteristics causing detrimental effects on survival after lung transplantation

The impact of Luminex-detected HLA antibodies on outcomes after lung transplantation is unclear. Herein we have undertaken a retrospective study of pre-transplant sera from 425 lung transplants performed between 1991 and 2003. Pre-transplant sera, originally screened by complement-dependent cytotoxicity (CDC) assays, were retrospectively tested for the presence of HLA-specific antibodies using HLA-coated Luminex beads and C4d deposition on Luminex beads. The results were correlated with graft survival at 1 year. Twenty-seven patients were retrospectively identified as having been transplanted against donor-specific HLA antibodies (DSA) and 36 patients against non-donor-specific HLA antibodies (NDSA). DSA-positive patients had 1-year survival of 51.9% compared with 77.8% for NDSA and 71.8% for antibody-negative patients (p = 0.029). One-year survival of patients with complement-fixing DSA was 12.5% compared with 62.5% for non-complement-fixing DSA, 75.8% for non-complement-fixing NDSA and 71.8% for antibody-negative patients (p < 0.0001). DSA-positive patients with mean fluorescence intensity (MFI) >5,000 had 1-year survival of 33.3% compared with 71.4% for MFI 2,000 to 5000 and 62.5% for MFI <2,000 (p = 0.0046). Multivariable analysis revealed DSA to be an independent predictor of poor patient survival within 1 year (p = 0.0010, hazard ratio [HR] = 3.569) as well as complement-fixing DSA (p < 0.0001, HR = 11.083) and DSA with MFI >5,000 (p = 0.0001, HR = 5.512). Pre-formed DSA, particularly complement-fixing DSA, and high MFI are associated with poor survival within the first year after lung transplantation. Risk stratification according to complement fixation or MFI levels may allow for increased transplantation in sensitized patients.

A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.

The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals’ physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.

Auto-antibody profiling with over 9,500 human expressed proteins (TECH1P.853)

Abstract Autoantibody profiling is a powerful method to probe the immune system for early signs of disease. However, progress has been limited by the availability of suitable antigens and high-throughput tools for screening large numbers of samples. Most antigens available have been produced in bacterial or insect cells and thus may not be optimal for autoantibody profiling. Although protein microarrays have been widely used to identify common autoantibodies in many diseases, creating smaller panels of antigens for larger screening studies has been difficult and costly. We have developed rapid methods for coupling proteins to Luminex beads allowing us to create and validate focused beads arrays in just a few days using native human proteins that have been expressed and purified in HEK293 cells. We have also developed a variable-stringency assay system that allows the user to optimize the detection specificity according to their particular application. Internal and external controls validate the presence of each protein and the IgG from the patient sample. Bead arrays for classic autoimmune antigens, as well as human breast, prostate, ovarian, pancreatic and lung cancer antigens have been prepared and custom arrays can be prepared from any of OriGene’s over 9500 purified proteins. Preliminary results with serum samples from traditional autoimmune patients, breast cancer, and Type II Diabetes support the utility of the approach.