Antibody Reactivity Patterns Research Articles

A panel of recombinant Leishmania donovani cell surface and secreted proteins identifies LdBPK_323600.1 as a serological marker of symptomatic infection.

Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.

PhIP-Seq Reveals Autoantibodies for Ubiquitously Expressed Antigens in Viral Myocarditis.

Simple SummaryMyocarditis is the inflammation of the heart muscle, and viral infections are a common cause of this disease. Myocarditis in some patients can progress to dilated cardiomyopathy (DCM). The mouse model of coxsackievirus B3 (CVB3) is commonly used to understand this disease progression in DCM patients. In this paper, we have attempted to analyze antibodies for heart antigens that could be produced as a result of heart damage in animals infected with CVB3 using a technique called Phage ImmunoPrecipitation Sequencing (PhIP-Seq). The analyses led us to identify antibodies for several proteins that were not previously reported that may have relevance to human disease.Enteroviruses such as group B coxsackieviruses (CVB) are commonly suspected as causes of myocarditis that can lead to dilated cardiomyopathy (DCM), and the mouse model of CVB3 myocarditis is routinely used to understand DCM pathogenesis. Mechanistically, autoimmunity is suspected due to the presence of autoantibodies for select antigens. However, their role continues to be enigmatic, which also raises the question of whether the breadth of autoantibodies is sufficiently characterized. Here, we attempted to comprehensively analyze the autoantibody repertoire using Phage ImmunoPrecipitation Sequencing (PhIP-Seq), a versatile and high-throughput platform, in the mouse model of CVB3 myocarditis. First, PhIP-Seq analysis using the VirScan library revealed antibody reactivity only to CVB3 in the infected group but not in controls, thus validating the technique in this model. Second, using the mouse peptide library, we detected autoantibodies to 32 peptides from 25 proteins in infected animals that are ubiquitously expressed and have not been previously reported. Third, by using ELISA as a secondary assay, we confirmed antibody reactivity in sera from CVB3-infected animals to cytochrome c oxidase assembly factor 4 homolog (COA4) and phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1), indicating the specificity of antibody detection by PhIP-Seq technology. Fourth, we noted similar antibody reactivity patterns in CVB3 and CVB4 infections, suggesting that the COA4- and PIK3AP1-reactive antibodies could be common to multiple CVB infections. The specificity of the autoantibodies was affirmed with influenza-infected animals that showed no reactivity to any of the antigens tested. Taken together, our data suggest that the autoantibodies identified by PhIP-Seq may have relevance to CVB pathogenesis, with a possibility that similar reactivity could be expected in human DCM patients.

HLA graph, a free and ready-to-use bioinformatics tool to explore anti-HLA eplets reactivity pattern.

HLA antigens are highly polymorphic, and their immunogenicity is dependent on the configurations of polymorphic amino acids. Monitoring anti-HLA immunization is essential in organ transplantation, as antibodies directed against HLA molecules are a major cause of rejection. Anti-HLA antibodies are not specific for HLA antigens, but recognize B-cell epitopes present on HLA molecules. To better understand antibody reactivity patterns, we calculated the Spearman correlation of the mean fluorescence intensity (MFI) of anti-HLA antibodies identified by a single-antigen assay performed using a Luminex® immunobeads assay on a large number of samples. Then, we built a computer tool analyzing antibody reactivity patterns with an accessibility by a web browser linked to the International Epitope Registry. We also extended our model to Onelambda® and Lifecodes® single-antigen class I and class II assays. The resulting MFI correlations reflect HLA antibody cross-reactivity and eplets similarity. We built HLA Graph, a computer tool that analyzes the eplets involved in antibody reactivity profiles. HLA Graph is usable with Onelambda® and Lifecodes® single-antigen class I and class II assays. The interpretation of reactivity against alleles not tested by the antibody assays and against the alpha and beta chains of HLA-DQ and HLA-DP loci were also developed. HLA Graph is a free and ready-to-use bioinformatics tool that can be used by all laboratories performing anti-HLA antibody identification by immunobead assay.

Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: Antibody dynamics and linkage to severity of COVID-19 in hospital inpatients

Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: Antibody dynamics and linkage to severity of COVID-19 in hospital inpatients

Distinct SARS-CoV-2 antibody reactivity patterns elicited by natural infection and mRNA vaccination

We analyzed data from two ongoing COVID-19 longitudinal serological surveys in Orange County, CA., between April 2020 and March 2021. A total of 8476 finger stick blood specimens were collected before and after a vaccination campaign. IgG levels were determined using a multiplex antigen microarray containing antigens from SARS-CoV-2, SARS, MERS, Common CoV, and Influenza. Twenty-six percent of specimens from unvaccinated Orange County residents in December 2020 were SARS-CoV-2 seropositive; out of 852 seropositive individuals 77 had symptoms and 9 sought medical care. The antibody response was predominantly against nucleocapsid (NP), full length, and S2 domain of spike. Anti-receptor binding domain (RBD) reactivity was low and not cross-reactive against SARS S1 or SARS RBD. A vaccination campaign at the University of California Irvine Medical Center (UCIMC) started on December, 2020 and 6724 healthcare workers were vaccinated within 3 weeks. Seroprevalence increased from 13% pre-vaccination to 79% post-vaccination in January, 93% in February, and 99% in March. mRNA vaccination induced higher antibody levels than natural exposure, especially against the RBD domain and cross-reactivity against SARS RBD and S1 was observed. Nucleocapsid protein antibodies can be used to distinguish vaccinees to classify pre-exposure to SARS-CoV-2 Previously infected individuals developed higher antibody titers to the vaccine than non pre-exposed individuals. Hospitalized patients in intensive care with severe disease reach significantly higher antibody levels than mild cases, but lower antibody levels compared to the vaccine. These results indicate that mRNA vaccination rapidly induces a much stronger and broader antibody response than SARS-CoV-2 infection.

Human Microbiota Flagellins Drive Adaptive Immune Responses in Crohn’s Disease

Crohn's disease and ulcerative colitis are characterized by dysregulated adaptive immune responses to the microbiota in genetically susceptible individuals, but the specificity of these responses remains largely undefined. Therefore, we developed a microbiota antigen microarray to characterize microbial antibody reactivity, particularly to human-derived microbiota flagellins, in inflammatory bowel disease. Sera from healthy volunteers (n= 87) at the University of Alabama at Birmingham and from patients recruited from the Kirklin Clinic of University of Alabama at Birmingham Hospital, including patients with Crohn's disease (n= 152) and ulcerative colitis (n= 170), were individually probed against microbiota bacterial flagellins of both mouse and human origin and analyzed for IgG and IgA antibody responses. Circulating flagellin-reactive T effector (CD4+CD154+) and T regulatory (CD4+CD137+) cells were isolated and evaluated in selected patients. Resulting adaptive immune responses were compared with corresponding clinical data to determine relevancy to disease behavior. We show that patients with IBD express selective patterns of antibody reactivity to microbiota flagellins. Patients with Crohn's disease, but not patients with ulcerative colitis, display augmented serum IgG to human ileal-localized Lachnospiraceae flagellins, with a subset of patients having high responses to more than 10 flagellins. Elevated responses to CBir1, a mouse Lachnospiraceae flagellin used clinically to diagnose CD, correlated with multi-Lachnospiraceae flagellin reactivity. In this subset of patients with CD, multi-flagellin reactivity was associated with elevated flagellin-specific CD154+CD45RA- T memory cells, a reduced ratio of flagellin-reactive CD4+ T regulatory to T effector cells, and a high frequency of disease complications. Patients with Crohn's disease display strong adaptive immune response to human-derived Lachnospiraceae flagellins, which may be targeted for prognosis and future personalized therapies.

Distinct SARS-CoV-2 antibody reactivity patterns in coronavirus convalescent plasma revealed by a coronavirus antigen microarray

A coronavirus antigen microarray (COVAM) was constructed containing 11 SARS-CoV-2, 5 SARS-1, 5 MERS, and 12 seasonal coronavirus recombinant proteins. The array is designed to measure immunoglobulin isotype and subtype levels in serum or plasma samples against each of the individual antigens printed on the array. We probed the COVAM with COVID-19 convalescent plasma (CCP) collected from 99 donors who recovered from a PCR+ confirmed SARS-CoV-2 infection. The results were analyzed using two computational approaches, a generalized linear model (glm) and random forest (RF) prediction model, to classify individual specimens as either Reactive or non-reactive against the SARS-CoV-2 antigens. A training set of 88 pre-COVID-19 specimens (PreCoV) collected in August 2019 and102 positive specimens from SARS-CoV-2 PCR+ confirmed COVID-19 cases was used for these analyses. Results compared with an FDA emergency use authorized (EUA) SARS-CoV2 S1-based total Ig chemiluminescence immunoassay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and with a SARS-CoV-2 S1-S2 spike-based pseudovirus micro neutralization assay (SARS-CoV-2 reporter viral particle neutralization titration (RVPNT) showed high concordance between the three assays. Three CCP specimens that were negative by the VITROS CoV2T immunoassay were also negative by both COVAM and the RVPNT assay. Concordance between VITROS CoV2T and COVAM was 96%, VITROS CoV2T and RVPNT 93%, and RVPNT and COVAM 91%. The discordances were all weakly reactive samples near the cutoff threshold of the VITROS CoV2T immunoassay. The multiplex COVAM allows CCP to be grouped according to antibody reactivity patterns against 11 SARS-CoV-2 antigens. Unsupervised K-means analysis, via the gap statistics, as well as hierarchical clustering analysis revealed three main clusters with distinct reactivity intensities and patterns. These patterns were not recapitulated by adjusting the VITROS CoV2T or RVPNT assay thresholds. Plasma classified by COVAM reactivity patterns offers potential to improve CCP therapeutic efficacy CoV2T alone. The use of a SARS-CoV-2 antigen array can qualify CCP for administration as a treatment for acute COVID-19, and interrogate vaccine immunogenicity and performance in preclinical, clinical studies, and routine vaccination to identify antibody responses predictive of protection from infection and disease.

Multipathogen Analysis of IgA and IgG Antigen Specificity for Selected Pathogens in Milk Produced by Women From Diverse Geographical Regions: The INSPIRE Study.

Breastfeeding provides defense against infectious disease during early life. The mechanisms underlying this protection are complex but likely include the vast array of immune cells and components, such as immunoglobulins, in milk. Simply characterizing the concentrations of these bioactives, however, provides only limited information regarding their potential relationships with disease risk in the recipient infant. Rather, understanding pathogen and antigen specificity profiles of milk-borne immunoglobulins might lead to a more complete understanding of how maternal immunity impacts infant health and wellbeing. Milk produced by women living in 11 geographically dispersed populations was applied to a protein microarray containing antigens from 16 pathogens, including diarrheagenic E. coli, Shigella spp., Salmonella enterica serovar Typhi, Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis and other pathogens of global health concern, and specific IgA and IgG binding was measured. Our analysis identified novel disease-specific antigen responses and suggests that some IgA and IgG responses vary substantially within and among populations. Patterns of antibody reactivity analyzed by principal component analysis and differential reactivity analysis were associated with either lower-to-middle-income countries (LMICs) or high-income countries (HICs). Antibody levels were generally higher in LMICs than HICs, particularly for Shigella and diarrheagenic E. coli antigens, although sets of S. aureus, S. pneumoniae, and some M. tuberculosis antigens were more reactive in HICs. Differential responses were typically specific to canonical immunodominant antigens, but a set of nondifferential but highly reactive antibodies were specific to antigens possibly universally recognized by antibodies in human milk. This approach provides a promising means to understand how breastfeeding and human milk protect (or do not protect) infants from environmentally relevant pathogens. Furthermore, this approach might lead to interventions to boost population-specific immunity in at-risk breastfeeding mothers and their infants.

172. Serum Igg Profiling Healthy 1- and 2- year Old Toddlers Reveals a Subgroup with Clinically Informative Reactivities to Pathogens and Autoantigens

BackgroundThe antibody repertoire in an infant/toddler develops in response to the microbiome, infections, environmental exposures, and vaccinations. Monitoring the specificity of these antibody responses in normal toddlers will provide indicators of disease susceptibility.MethodsThe serum Immunoglobulin (Ig)G and IgM antibody reactivity patterns in 1- and 2-year-old healthy toddlers was determined by examining the Ig specificity to diverse infectious agents, autoantigens and vaccine antigens with an antigen array. The toddlers were stratified based on their antibody reactivity to these diverse antigens with a normalized fluorescence intensity measure. Repeat profiling was performed at year 2 to reveal longitudinal changes in the IgG and IgM responses. Clinical information, along with DNA sequencing, and selected cytokine assays were used to establish an odds ratio for immune disease potential among the cohort.ResultsHealthy 1- and 2- year old IgG responses revealed cohorts of low, moderate, and high Ig responder groups that was unconnected with total serum IgG levels. The high responder group had elevated IgG reactions to selected pathogens, particularly viruses as well as to autoantigens. This high reactivity group, representing 17% of the cohort, had high odds ratios with maternal gestational diabetes, age, and a family history of asthma. While all toddlers developed strong antibody responses to Measles-Mumps-Rubella vaccines (MMR), more variation was noted towards other vaccines. In infections to Molluscum contagiosum, the IgG serum levels were transient regardless of the responder group. The high responder group had DNA polymorphisms linked to enhanced immune responses that correlated with elevated cytokine levels as well as eczema and asthma. A subset of toddlers has strong IgG responses to pathogens and vaccines ConclusionA subset of normal healthy toddlers has a high potential for immune system abnormalities and autoimmunity based on higher serum antibody responses to pathogens and autoantigens, genetic polymorphisms, and elevated cytokine responses.Disclosures All Authors: No reported disclosures

Antibody-Mediated Rejection Due to Donor-Specific HLA-DQB1 and DQA1 Antibodies After Kidney Transplantation: A Case Report

BackgroundDonor-specific HLA antibody (DSA) is associated with the risk of allograft loss due to antibody-mediated rejection (ABMR). The majority of de novo DSA after kidney transplantation is directed toward donor HLA-DQ antigens. A HLA-DQ antigen is a heterodimer consisting of an alpha and beta chain. Traditionally, HLA-DQA1 typing has not been part of the pretransplant evaluation. Therefore, DQ alpha proteins are not usually taken into account in the interpretation of HLA-DQ antibody reactions. MethodsWe hereby present a case of a kidney transplant recipient with 0% pretransplant panel reactive antibody. She received kidney allograft from her husband. Two years after transplantation, she experienced abdominal swelling, and enlargement of transplanted kidney was identified. A biopsy of the allograft kidney demonstrated chronic active ABMR. DSAs were investigated using immunoglobulin G (IgG) and C1q single antigen bead (SAB) assay. HLAMatchmaker analysis was performed to identify eplets that explain the antibody reactivity patterns. ResultsThe IgG SAB analysis of a patient’s serum at the time of rejection showed positive reactions with all DQ2-carrying beads with mean fluorescence intensity (MFI) > 10000. However, the C1q assay demonstrated strong reaction to only HLA-DQA1∗05:01-DQB1∗02:01-carrying bead with MFI = 22462, whereas weak or no reactions against other HLA-DQ2-carrying beads were found. High-resolution HLA typing revealed that HLA-DQA1∗05:01 and DQB1∗02:01 were mismatched donor antigens. HLAMatchmaker analysis showed that the antibodies were reactive toward 40GR3 eplet on DQA1 and 45GE3 eplet on DQB1. ConclusionsThis case highlights the clinical significance of antibodies specific to both DQ alpha and DQ beta chains after kidney transplantation.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor‐specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA‐specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA‐DR‐specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA‐DR antigen‐reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA‐DRB1*01:01, HLA‐DRB1*04:01, and HLA‐DRB1*07:01 antigen‐reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA‐DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Characterisation of ACP5 missense mutations encoding tartrate-resistant acid phosphatase associated with spondyloenchondrodysplasia.

Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism.

Novel variants of infectious bursal disease virus can severely damage the bursa of fabricius of immunized chickens

In recent years, atypical infectious bursal disease (IBD) with severe immunosuppression has brought new threats to the poultry industry and has caused considerable economic losses. Novel variant infectious bursal disease virus (IBDV) has been identified as the etiological pathogen and for unknown reasons is widespread in poultry on many chicken farms in China that have been immunized with vaccines against very virulent IBDV (vvIBDV). Using immunoprotection experiments in specific-pathogen-free chickens, we first verified that novel variant IBDV could severely damage the bursa of Fabricius of the important immune organ of immunized chicken in the presence of antibodies induced by three types of vvIBDV vaccines, which is a primary reason for the current epidemic of atypical IBD. Monoclonal antibody reactivity patterns and cross-neutralization assays further confirmed the obvious antigenic mismatch between novel variant IBDV and vvIBDV. Sequence analysis of the genome of novel variant IBDV (SHG19 strain) was performed and the key amino acid residues that might be involved in antigenicity and virulence differences of novel variant IBDV compared to vvIBDV were further analyzed. This study not only determined the primary reason for the atypical IBD epidemic, but also remind us of the urgency for developing new vaccines against novel variant IBDV.

Anti-HLA Antibody: The Role of Epitopes in Organ Transplantation.

Recent developments have been achieved for better matching in solid-organ transplantation by epitope matching. HLA matching remains a standard immunologic strategy to determine organ compatibility for recipients. Advancements in tissue typing have introduced HLA matching at the epitope level. For this, B cells are able to recognize polymorphic amino acid configurations, which are named epitopes. However, antibody production against these epitopes may be a leading cause of rejection. Although data to support the added clinical benefit of epitope matching over traditional antigen matching are still lacking, there are at least 2 theoretical reasons for epitope matching: (1) to avoid future allosensitization with the development of anti-HLA antibodies and (2) to allow selection of a suitable allograft for highly sensitized patients through virtual crossmatch. Sensitive antibody tests with a comprehensive panel of single alleles have yielded informative, donor-specific antibody reactivity patterns that can be analyzed with a computer algorithm. This program (HLAMatchmaker) uses structurally defined eplets to describe epitopes that can react with specific antibodies. Although low mean fluorescence intensity levels by Luminex (Austin, TX, USA) assay are usually considered low risk for solid organ transplant, it could be important in posttransplant humoral rejection. Thus, specific shared eplets should always be investigated with respect to previous transplant mismatches. Epitopes are present on a single HLA (private epitope) or shared by multiple antigens (public epitope). The phenomenon of crossreactivity in HLA testing, often explained as crossreactive groups of antigens with antibody, can be clearly explained now by public epitopes, an antibody targeting an epitope that shows positive reaction with all antigens sharing the epitope. A better understanding of the immunogenicity and structural characteristics of HLA epitopes will guide us to consider epitope matching as an important parameter for donor selection in kidney transplant in the near future.

The prevalence of antibodies against the HLA-DRB3 protein in kidney transplantation and the correlation with HLA expression.

Human leukocyte antigen (HLA)-DRB3 is a functional HLA class II gene, which has a limited allele diversity in the human population. Furthermore, the HLA-DRB3 gene is only present in a subset of individuals. Therefore, in organ transplantation, this HLA molecule is frequently mismatched between patient and graft donor and thus antibodies against this mismatched HLA molecule can develop. In this study, we aimed to evaluate the prevalence and reactivity of these antibodies and aimed to identify factors that underlie antibody formation against HLA-DRB3. We showed in our patient cohort that HLA-DRB3 antibodies are identified in about 7% of all patients that were screened with solid phase assays. In these assays, we observed multiple antibody reactivity patterns indicating that HLA-DRB3 harbours multiple epitopes. In those cases, where we succeeded at tracing back the induction of these antibodies to the molecular HLA typing of the immunogenic event, we noticed a different frequency of HLA-DRB1 allele groups in the donors as compared to a control group. To a certain extent this distribution (e.g. HLA-DRB1*11 individuals) could be linked to an altered expression level. However, it also appears that different HLA-DRB3 alleles (e.g. HLA-DRB3*01 group) vary in their immunogenicity without having an expression difference. In conclusion, our study provides information on the immunogenicity and reactivity patterns of antibodies against HLA-DRB3 in kidney transplantation, and it points towards the possibility of HLA expression as a factor underlying antibody formation.

Immunoprofiling of Chlamydia trachomatis using whole-proteome microarrays generated by on-chip in situ expression

Using Chlamydia trachomatis (Ct) as a complex model organism, we describe a method to generate bacterial whole-proteome microarrays using cell-free, on-chip protein expression. Expression constructs were generated by two successive PCRs directly from bacterial genomic DNA. Bacterial proteins expressed on microarrays display antigenic epitopes, thereby providing an efficient method for immunoprofiling of patients and allowing de novo identification of disease-related serum antibodies. Through comparison of antibody reactivity patterns, we newly identified antigens recognized by known Ct-seropositive samples, and antigens reacting only with samples from cervical cancer (CxCa) patients. Large-scale validation experiments using high-throughput suspension bead array serology confirmed their significance as markers for either general Ct infection or CxCa, supporting an association of Ct infection with CxCa. In conclusion, we introduce a method for generation of fast and efficient proteome immunoassays which can be easily adapted for other microorganisms in all areas of infection research.

Helicobacter pylori serological biomarkers of gastric cancer risk in the MCC-Spain case-control Study

BackgroundHelicobacter pylori infection is one of the main risk factors for non-cardia gastric cancer. However, only a minority of infected persons develop the disease. This study aims at identifying H. pylori related serological biomarkers of risk for gastric cancer. MethodsIncident gastric cancer cases and population controls (age, sex and region frequency-matched) from the MCC-Spain multicase-control Study were included. Seroreactivities against 16H. pylori proteins were determined using multiplex serology. Infection was defined as seropositivity against≥4 proteins. Relation of serological results to non-cardia and cardia gastric cancer was assessed using multivariable mixed logistic regression and principal components analysis. ResultsSeroprevalence was 88% among 2071 controls, 95% among 202 non-cardia gastric cancer cases (OR=1.9 (95% CI: 1.0–3.6)) and 85% among 62 cardia cancer cases (OR=0.5 (95% CI: 0.3–1.1)). In infected subjects, seropositivity for UreA, HP231, NapA and Cagδ was associated with lower non-cardia gastric cancer risk, while seropositivity for CagA and VacA was associated with higher risk. Seropositivity for CagA and seronegativity for Cagδ maintained the association after additional adjustment by serostatus of significant proteins. We identified two antibody reactivity patterns: the “virulent-pattern”, related to a threefold higher risk of non-cardia gastric cancer and the “non-virulent pattern”, related to a 60% decreased risk (4th vs. first quartile). ConclusionsIn our population, people seropositive for H. pylori were characterized by two patterns of antibody reactivity against H. pylori proteins: 1) Combined high seroreactivity against several proteins, associated with a lower non-cardia gastric cancer risk, and 2) High seroreactivity against CagA and VacA, associated with an increased risk.

False positive antibody specificities in the One Lambda LABScreen® single antigen assay can be identified using surrogate crossmatch and the One Lambda LABScreen® mixed bead assay

The One Lambda LABScreen® Single Antigen assays (LS1A04 and LS2A01) have developed antibody reactivity patterns suggestive of false positive reactivity. This study was performed using patient sera whose positive One Lambda LABScreen® Single Antigen (LSA) reactivity was found to be discrepant with negative or weak positive One Lambda LABScreen® mixed bead (LSM) assays performed during routine monthly testing. The aim of this study was to determine how reliable the LSM assay is at detecting false positive LSA reactivity and which patterns of reactivity in the LSA assay are false positive. 107 patient sera were suspected of having false positive specificities (>2000 MFI) using the LSA assays. These sera were also screened using the LSM assay. Sera that were negative or weak positive in the LSM were crossmatched using a total of 134 surrogate donor cells due to some sera containing more than one suspected false positive specificity. In 5 cases, the One Lambda mixed bead assay was positive but a surrogate crossmatch was performed due to the suspicious nature of the antibody reactivity pattern in the single antigen assay. Of the 107 discrepant samples, 102 were negative in surrogate crossmatch and also negative/weak positive in the LSM assay. 5 sera tested positive in surrogate crossmatch, but only 1 of these sera tested negative in the LSM assay. The other 4 sera tested positive in both the LSA and LSM assays. Conversely, there were 5 sera that were positive in both the LSA and LSM assays, but were negative in surrogate crossmatch. Based on this data, when there was a suspected false positive specificity or pattern in the LSA, the LSM assay was false positive only 4.6% of the time. Another benefit of this study was the identification of common false positive specificities and reaction patterns which include the following: specificities: A1, A2, A23, A24, A30, A31, A36; B8, B37, B44, B45, B63; DR53; DQ2(DQA1∗05:01/DQB1∗02:01); DQB1∗06:03 patterns: B57 and B58; Cw1, C∗12, C∗15; DR16 and DRB1∗04:04; DQ7, DQ8, and DQ9. The positive LSA assay, in the presence of a negative or weak positive LSM result, was false positive 95% of the time. The LSM assay is a reliable method for detection of false positivity in the LSA assay.

P237 Specific pattern of non-specific ANTI-HLA antibody reactivity?

Solid phase assays (SPAs) have significantly improved our ability to assess the presence of anti-HLA antibody (Abs) that would be deleterious to the allograft post-transplant. SPAs have also demonstrated increased specificity and sensitivity for alloantibody detection. However, several studies have identified intrinsic and extrinsic factors that negatively impact the reliability of SPAs. Thus, substances in sera that inhibit/interfere with the Abs binding have been identified. In addition, presence of denatured HLA antigens and/or inconsistent antigen density on latex beads has been noted. These factors coupled with the increased sensitivity of the SPAs could lead to false positive/false negative results. In the present study, using single antigen beads (SAB), we demonstrate the detection of a highly specific pattern of HLA Class II alloantibodies (MFI > 1,000) against a small subset of DRB1, DRB3 and DP antigens. What is remarkable about this pattern is that a near identical profile of alloantibodies was detected in at least 6 patients (5 pre- and 1 post-transplant) and was conserved over time. Thus, Abs against the following antigens (ranked from highest MFI to lowest MFI; DR52 (03:01) – DR14:02 – DP10 – DR16:01 – DR8 - DR14:54 – DR9 – DR16:02 – DR14:01 – DP13 – DP19 – DR13) were identified in all the patients. An epitope that would help explain this pattern of reactivity could not be identified. To confirm this pattern, the sera were tested using SAB from another vendor. This pattern was completely absent, suggesting that the observed reactivity was non-specific. However, pretreatment of sera with a variety of products known to reduce interfering substances did not alter the reactivity pattern suggesting that it was “real”. Testing of sera for the presence of HLA Class I and II Abs using screening beads (each coated with multiple HLA antigens) confirmed the observations made with SAB further suggesting that the observed reactivity was “real”. Finally, surrogate flow crossmatches with donor cells expressing one or more corresponding antigens were largely T- and B-cell negative suggesting that the observed reactivity was most likely non-specific (surreal?). This study underscores the complexity involved in interpreting and then assigning presence/absence of anti-HLA alloantibodies using the highly sensitive SPAs.

P195 Single antigen dilution reveals interference and falsely low MFI for donor specific antibodies

A 48 year old man presented for second kidney transplant with a potential living donor, his brother. His first transplant was in 2000. At evaluation, the patient had HLA single antigen class I and II testing on neat serum treated with DTT, flow crossmatch (FCXM, ±Pronase) and cytotoxic crossmatch (CDC, ±DTT). Neat serum showed antibodies to HLA-A29 and B51, both repeat mismatches from his prior transplant, and an odd pattern of DQA1 antibodies (Table 1). The initial FCXM with his brother was positive on T (187MCS) and B cells (408MCS). The B CDC was also positive. The patient displayed DSA to HLA-A29 (8277MFI), B51 (1588MFI), and DQA1∗02 (8467MFI) in the same serum. However, the crossmatch was unexpectedly strong given the MFI. Importantly, the patient’s first transplant was from another brother, who was HLA identical to this potential sibling donor. We suspected that the patient in fact had strong antibodies to HLA-DQ2 and DQ7, which were repeat mismatches. It was curious that the beads with DQA1∗05 had the lowest MFI as this combination was exactly that present in the first donor. In addition, there were apparently allele-specific DQA1∗03:01 antibodies, which is unusual. Thus, we performed dilution of the serum at 1:10 and 1:100 to determine whether there was interference with very strong antibodies. Dilution of the patient’s serum resulted in a significant increase in MFI for DQ beads. For example, DQA1∗03:02/DQB1∗03:02 went from 944MFI to 10,533 MFI at 1:10. Dilution also showed that the patient’s DSA was to HLA-DQ2 and DQ7 rather than to DQA1 alleles. The donor was declined and paired exchange was recommended. We also noted on the single antigen reports that the patient exhibits “prozone” and the antibodies to DQ7 and DQ2 are much stronger than represented by the MFI in neat serum. This case demonstrates the utility of serum dilution to identify strong antibodies with falsely low MFI, and illustrates a pattern of antibody reactivity that should appear “suspicious” for interference: for example, apparent DQA1 allele-specific antibodies.Download : Download high-res image (208KB)Download : Download full-size image