In the event of a radiological or nuclear disaster, there will be a need for therapies that can mitigate the acute hematopoietic and gastrointestinal (GI) radiation syndromes expected to develop in many of the victims of the disaster. Currently, there are FDA approved G-CSF and GM-CSF agents for mitigating the myelosuppression associated with the hematopoietic syndrome. However, an effective mitigation agent for acute GI syndrome is currently missing from our clinical armamentarium. Basic FGF (FGF2) benefits both GI and hematopoietic
syndromes in mouse models. However, natural FGF2 is severely depleted by total-body irradiation in humans, and replacement with hrFGF2 has logistical challenges and production costs that make clinical use and strategic stockpiling unrealistic. We hypothesize that FGF-P, a small FGF2 mimetic peptide, mitigates acute radiation-induced GI syndrome through a variety of cooperating mechanisms, including decreased mucosal cell loss, improved proliferation of small bowel mucosa and gut barrier function, and reduced bacterial translocation. It also helps maintain progenitor cells through signaling pathways that mimic natural FGFs. Our current preclinical studies of FGF-P in rodent survival and mechanistic studies are demonstrating these expected benefits, including improved GI barrier function, electrolyte transport, and mitogenesis for at least 30-day post-irradiation. This is resulting in increased survival time and the number of survivors when FGF-P is given subcutaneously beginning 24 hours post radiation exposure. In an effort to more fully understand the mechanisms by which FGF-P is mitigating GI-ARS we are examining a number of pro- and anti-inflammatory cytokine and chemokine beginning prior to irradiation, within the first 10 days following administration of FGF-P to 30 post-irradiation. The cytokine and chemokine studies are being conducted with the use of the Bio-Rad Bio-Plex Pro 23-plex Mouse and Rat cytokine kits, and 33-plex mouse chemokine kit, run on a Luminex 200 instrument. These multi-plex assay systems provide us with the needed breadth of cytokine and chemokine coverage for our mechanistic studies while minimizing the volume of plasma sample needed to perform the assay. From the results of the cytokine and chemokine studies in the rodent models, we will be able to identify common markers of efficacy of the FGF-P in the rodent models that could be used to translate the results of these pre-clinical studies to higher animal models and ultimately into humans as we advance our drug development efforts with the goal of gaining FDA approval of the use of FGF-P as a mitigator of GI-ARS using the Animal Rule.

Biologic based therapeutics are used for treatment of many diseases and represent the most advanced therapies available. These drugs often generate immune responses in patients that may impact drug efficacy and adversely affect patient safety. Regulatory agencies responsible for drug approval recommend an assessment of anti-drug immune responses including a screening assay to detect the presence of anti-drug antibodies (ADA), titer, assessment of drug neutralizing activity, and classification of ADA responses with respect to antibody class and subclass. The industry uses singleplex assays to detect and characterize ADA responses in a tiered sequential manner. This presentation will introduce the concepts of Immunogenicity and share a novel multiplexed based approach to detect and characterize ADA responses.

We selected Humira, a human IgG1 antibody that binds to TNFa as the biologic drug for this study which has been shown to induce ADA responses. Biotinylated Humira was used to capture and isolate ADA from clinical samples using streptavidin coated plates. This also removed non-specific antibodies and other matrix components from the purified ADA preparation. The ADA were subsequently mixed with Luminex bead sets each coupled to an Fc specific anti-human Ig class or subclass to delineate the different classes and subclasses from each other. These included human IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE. The beads were washed and incubated with biotinylated Humira to detect drug specific ADA captured on each bead set. Streptavidin-phycoerythrin was added followed by analysis of the bead sets in a Luminex Flexmap-3D instrument. The median fluorescence intensity (MFI) of >50 beads per set was calculated and used in the final data analysis.

The data show that each isotype specific bead set had good specificity for the respective isotype. The detection of ADA in samples was based on a statistically derived cut point based on a set of 50 drug-naïve samples analyzed in the assay 6 times across several days. This cut point was set so that 95% of drug naïve samples fall below the cut point and samples with drug specific antibodies present would generate a signal above the cut point. The cut point was set to yield a 5% false positive rate to ensure all samples with ADA would be detected. A separate cut point was calculated for each specific antibody class or subclass. Clinical samples from psoriasis patients and other autoimmune disease patients were analyzed for the presence of ADA. The results show the presence of ADA and a mixture of different isotypes present. The sensitivity of the assay was determined to be less than 50 ng/ml using a human anti-Humira antibody as a positive control. The presentation will also show data from clinical samples with respect to the presence of ADA and the respective isotypes present.

The multiplexed approach described here demonstrate an advantage over single assay approaches with respect to minimizing clinical sample freeze thaws, cost and labor, while providing a robust and reliable method for ADA evaluation. This method yielded results that were comparable to the FDA guidelines for assessment of Immunogenicity.

Background and aim:
Multiplexing of biomarker panels by microsphere-based technologies are robust and cost-effective methods. The QuantiGene™ Plex Assay (ThermoFisher Scientific) platform quantitates mRNA expression from crude lysates discriminating up to 80 target sequences. We aim at using expression analyses fo Human Papillomavirus (HPV) oncogenes and cellular biomarkers corresponding to cervical dysplasia progression for screening and diagnostic molecular assays. Currently used DNA-based HPV tests are highly sensitive for infection but have low specificity for true disease. This is especially problematic in LMIC like in Africa where HPV prevalence is high, no screening approaches exist, and healthcare systems are overburdened.

Methods:
A 40-plex QuantiGene™ assay was designed to quantify mRNA expression of the E7 oncogene of 18 high-risk HPV genotypes, cellular housekeeping genes for normalization, and 18 well established cellular biomarkers like proliferation markers, tumor suppressors, cancer stem cell transcription factors, and invasive tumor markers. Initially, the lysis procedure, sensitivity and specificity of the target sequences were tested in a pilot assay for feasibility with established cervical cancer cell lines. The suitability of the target panel and the assay procedure was tested on 1400 consecutive prospectively collected clinical samples. By logistic regression and ROC analyses marker sub-panels and cut offs were determined identifying and discriminating the different dysplastic stages and cervical cancer. Costs and workload were compared to a qRT-PCR-based method. The assay was used in two cervical cancer screening studies and compared to standard screening methods.

Results:
Feasibility of the assay was investigated by using established cervical cancer cell lines showing that HPV E7 and cellular biomarker expression was sufficiently sensitive detecting less than 40 cells. Most informative
biomarkers were identified by logistic regression. Characteristic marker panels for the dysplastic stages of cervical intraepithelial neoplasia (CIN)2+, CIN3+, and invasive cervical carcinoma were identified. High-grade dysplasia was detected in clinical smear samples with an accuracy of 90%. The comparison to qRT-PCR showed an acceptable correlation of results with remarkable lower costs and efforts for QuantiGene procedures. Using this assay in screening studies in Ethiopia and Germany resulted in much higher specificity for the detection of true dysplasia than the current standard of care. Importantly, work up of samples is simple (no nucleic acid purification and amplification), robust and especially adequate for
low-tech labs as found in LMIC.

Conclusion:
mRNA quantification by multiplexed bead-based technology is feasible in a cost effective way, simple and robust. Additionally, it presents an attractive means for IVD development for assay systems in cervical cancer screening and research.

The detection and quantification of human antibody levels directed against specific antigens is of utmost importance in several fields ranging from healthcare diagnostics, vaccine development, and immunological research to analysis of disease mechanisms. We have developed a serological xMAP® approach (xMAPr), which is superior over a classical one-stage dilution approach by generating high-dynamic range (HDR) data, by dramatically increasing in the dynamic range of over three magnitudes of order. This methodology was used in a population-based setting (SHIP cohort, N=996) to analyze different antibody isotype responses directed against 80 antigens derived from Staphylococcus aureus. The results were analyzed in  combination with different phenotypes and other Omics data revealing new insights in the S. aureus-specific immune response in healthy individuals.

Background and aim:
Worldwide malaria cases have declined. However, despite recent advances, resistance to front line drugs and insecticides means the burden remains high (219 million cases in 2017). The World Health Organisation’s (WHO) Global Technical Strategy (GTS) milestone for 2020 was to eliminate  malaria from 10 countries. One of the three main aims towards achieving this was to “Transform malaria surveillance into a core intervention”. However, declining malaria also means increasing difficulty in detecting infections. Therefore, as countries approach elimination, additional measures are required to improve the sensitivity of surveillance. Antibodies are a sensitive marker of exposure that are acquired after each infection and lost over time. By focusing on markers of recent exposure, we have developed a serosurveillance panel and a prototype lateral flow device to help contribute to malaria elimination efforts.

Methods:
We have developed a number of novel malaria species-specific recombinant antigens and coupled these to MagPlex® Microspheres, or “beads”, to develop a novel multiplex serology assay. Using the MAGPIX® platform, we have screened serum samples or eluted dried blood spots to determine prevalence of exposure.

Results:
We have generated a rich dataset from malaria-endemic regions spanning a wide geographical range and contrasting endemicities, totaling >35,000 serum samples (and dried blood spots) since 2017. In addition, we have established the MAGPIX® platforms in most of these sites to aid sample access and knowledge transfer.

Conclusion:
Using the MAGPIX® platform, we have developed a unique panel of reagents to help detect exposure to three of the main causes of human malaria, Plasmodium falciparum, P.vivax and P.knowlesi.

Background and aim:
Immune response must be measured in highly controlled conditions for reproducibility. Furthermore, whereas the cellular response plays a major role, only antibody production is assessed in most cases. For the purpose of determining responsiveness in ruminants, we developed a custom 15-plex cytokine/ chemokine assay with MERCK-Millipore.

Methods:
To evaluate the bovine cellular response, two approaches were used. Whole blood samples were collected from dairy cows, and immediately stimulated during 24 hours with heat-killed bacteria, TLR ligands, or soluble anti-CD3/CD28 monoclonal antibodies. A sample without stimulation was assayed in parallel. In another experiment, the response to an intravenous injection of ultrapure LPS was measured in plasma for the 24 hours post-injection. Bovine clones were used to reduce genetic variability.

Results:
Using ANOVA and data mining methods, we showed that the protocol is able to detect differences amongst conditions. For in vitro assays, the response varies according to the bacteria species, with S. uberis being the most potent stimulus, compared to S. aureus and E. coli that were distinct but more closely related to each other. Similarly, TLR ligands could be distinguished, and Gardiquimod was significantly different from the TLR2/4-activating ligands. LPS and E. coli provided very similar profiles confirming previous data indicating that LPS signaling mediates a large part of the E. coli response. Furthermore, when LPS was injected IV, plasma concentrations of cytokines and chemokines varied in parallel with the development of clinical signs and differed according to age and genetic background.

Conclusion:
We showed that the assay is able to reliably detect production of bovine cytokines and chemokines. High variability of the responses indicates that both environmental and genetic factors control the cellular response. Cytokine profiling is achievable for evaluation of the responses to infectious diseases and immunomodulation in cattle.

In order to emit less contaminants into the environment, and at the same time produce healthier foods that are free of those contaminants, all production systems should be investigated, i.e. whether improvements can be made. Within the Food-Agro industry, pesticides that are harmful to bees and insects are of concern and an example of such environmental contaminants. Not only because they cause a direct decrease of biodiversity, but also crop loss due to decreased pollination, which has a direct economic impact. Another actual example is the presence of antibiotics in dairy products. As both, the use of antibiotics and their presence in the food chain are related to antibiotic resistance of bacteria, complicating treatments for infectious diseases.

In the Biosensors & Bioassays department at Wageningen Food Safety Research (WFSR, formerly known as RIKILT Wageningen UR) a wide range of detection formats are explored for the development of food and feed safety, food fraud, food authenticity, food allergy and environmental contamination immunoassays.

There is a strong focus on the development of xMAP based assays on the MAGPIX® planar array analyzer. The easily transportable MAGPIX® analyzer can be taken from the laboratory into the field and implemented for portable on-site analysis. The point of need testing can be carried out in a simple laboratory set-up e.g. the back of a car. This can be of great importance since raw materials for food production can already be pre-screened prior to entering the food and feed production or a supply chain. Within WFSR, the xMAP technology is used for the multiplex detection of many different targets. This ranges from proteins, e.g. allergens, growth promotors, and fraud proteins, to very small molecules like mycotoxins, antibiotics, pesticides, tranquilizers, marine toxins and antiparasitics. The detection assays developed for these target analytes, and target analyte groups, cover a wide range of different food and environmental matrices, e.g. cereals, milk, honey, plants and water. Additionally, the xMAP technology is used to build single assays for high through put on-site analysis, e.g. the detection of fipronil in manure and eggs. Recently, a 13-plex for antibiotics was developed and validated for the detection of over 80 antibiotics in e.g. milk, urine and (drink) waters. However, in some cases, the availability of antibodies seriously hampers the development of a multiplex assay. This is especially the case for pesticides (>1000), where only a limited amount of antibodies are available. This complicates the development of broad range pesticides screening assays. Despite that, dedicated xMAP assays focusing on certain classes of pesticides are feasible and we recently started to work on the development of xMAP assays that focus on pesticides that have serious detrimental effects on bees.

In all the relevant fields of food safety and environmental research, biosensor methods are developed at WFSR using xMAP technology. It is considered as a fast and robust standard for ultiplexing, which is highlighted by combing previously developed unique multiplex assays for contaminants and animal drugs for different matrices. The xMAP technology is further used for benchmarking other multiplex technologies and newly developed prototypes of multiplexing equipment.

Background and aim:
Rapid and complex diagnosis of viral, bacterial and parasitic infections is highly desirable not only in clinical practice, but also in food safety or early warning systems. Multiple Oligonucleotide Ligation followed by
PCR (MOL-PCR) is easy to set up and allows to simply add detection targets to the existing panels. The MOL-PCR technique was used in the development of the panels for the detection and identification of viruses and bacteria originating from food and feed, viral pathogens of swine, bacterial pathogens in small ruminants and biothreat agents.

Methods:
MOL-PCR is based on the ligation of specific oligonucleotides in the presence of a specific target DNA. In the case of RNA viruses, reverse transcription is required. The ligation product serves as a template for the amplification with universal primers, one of which is labelled, in subsequent PCR. Visualization of PCR products is mediated by binding to magnetic fluorescent beads in a suspension array and is performed on a
MAGPIX® (Luminex) instrument. Each panel contained its own internal amplification control, which served to the differentiation of truly negative and false negative samples and control of presence of inhibitors. All the panels underwent the validation procedure adopted from the guidelines for the validation of the qPCR assays developed by FDA.

Results:
The result is a total of 5 validated panels for rapid detection and identification of the desired agents. The first includes viruses that are associated with food and water contamination - noroviruses, hepatitis A and E virus, rotaviruses and adenoviruses. The second panel is able to detect food-derived bacteria - Salmonella, Yersinia, Cronobacter, or pathogenic E. coli. Next panel is designed to detect viral agents in pigs such as African swine fever or various types of influenza. Panel dedicated to the small ruminants covers M. a. subsp. paratuberculosis, C. pseudotuberculosis, M. bovis and M. tuberculosis complex members - agents responsible for the tuberculosis and pseudotuberculosis infections. The last panel is developed for the needs of security forces and detects and typifies biothreat agents -B. anthracis, Y. pestis, F. tularensis and Brucella spp.

Conclusion:
The developed panels have been validated for complex diagnostics of clinical material, food including water, animals and other relevant matrices. The process of optimization of MOL-PCR was established and critical points, which require a special attention in the optimization were identified. Development of all the panels was targeted with the intention to fulfill the demands placed on the diagnostic tools.

Histone post-translational modifications (PTMs) play pivotal roles in chromatin dynamics and function, with alterations in the healthy profile associated with diverse human pathologies. The regulation and genomic distribution of PTMs is frequently studied by antibodies, despite the wide suspicion that a significant proportion of these reagents have specificity issues. To address this issue we have developed a high -sensitivity and -throughput approach that leverages the multiplexing ability of xMAP technology. We have created a library of >100 biotinylated PTM-containing designer nucleosomes (dNucs) that can be easily combined with Avidin-coated MagPlex beads and determined the binding profiles of >400 commercial ‘PTM-specific’ antibodies. Our results identify truly capable reagents to a large number of PTM targets, but also provide direct evidence that many antibodies in widespread use are highly cross-reactive. As such the current gold-standard screening approaches (immobilized histone PTM-peptide arrays) have proven poorly predictive of alternate assay platforms. In contrast Luminex assays with PTM-nucleosome substrates can be used to identify antibodies with true Chromatin Immuno- Precipitation (ChIP)-grade potential: this will be integral in creating new reagents to study these disease-relevant targets.

Emerging viral infections represent a major threat to the global health and their early detection may help anticipate outbreaks, improve patient’s care and plan appropriate Public Health level intervention strategies.
However, simultaneous serological detection of multiple viral infections is not amenable to routine utilization with conventional methods such as ELISA and Western blot. Our laboratory is investigating virus spillover at the human/wildlife interface in equatorial Africa with a specific focus on HIV/SIV (Human/Simian Immunedeficiency Viruses) and ebolaviruses. To identify the animal reservoir of ebolaviruses and to detect additional SIVs, to detect potential new cross-species transmissions from wildlife to humans and to anticipate future outbreaks, we developed in-house xMAP methods using Luminex® microspheres on BioPlex200 to screen for SIV and
ebolaviruses from diverse wildlife and Man in Africa. The xMAP technology has changed our way of hunting viruses in samples available, for some, at minute amount. xMAP helped us identify new viruses and helped us showing evidence of persistent cross-species transmissions of retroviruses in Africa.

This project is now running for ten years. The talk will share with the xMAP community our findings on retrovirology (HIV/SIV), on filoviruses (ebolavirus) and beyond.

Background and aim:
Glycosylation is an essential posttranslational modification used primarily for cellular communication. The formation of complex glycans as secondary gene products is dependent on the activity of various glycosyltransferases (GTs). Mutated GTs, however, can cause significant alterations in the glycan patterns expressed on cancerous cells, making those patterns potent biomarkers. Whereas the detection of these tumor-associated carbohydrate antigens (TACA) require primary tumor tissue, autoantibodies (AAbs) against cancer-specific glycans in human plasma can be detected with a simple and minimal invasive technique.

Methods:
The Glyco-Multiplex assay enables the simultaneous detection of AAbs against (i) various glycans with (ii) minor amounts of plasma from (iii) a large number of patients in (iv) a short time. For this approach, multiple, potentially tumor-associated, glycans are coupled to distinct Luminex® bead regions. The unique color-coding of the beads allows the measurement of an individual’s isotypespecific AAb profile against the selected glycans simultaneously in one single well. The adaption of the coupling protocol for the use of the KingFisherTM Flex enables the upscale of this assay to up to 96 different regions with negligible additional hands-on time.

Results:
A protocol for the coupling of glycans to carboxylated MagPlex® beads was established and optimized. Subsequently, suitable combinations of plasma concentrations and Ig-specific (IgM, IgG and IgA) detection antibodies and assay controls were evaluated to detect α-TACA AAbs in human plasma samples. To get a first impression on α-TACA AAb signals, plasma samples from colon cancer patients, lung cancer patients and healthy donors were measured using the Glyco-Multiplex. In accordance with literature, (auto)antibodies in both healthy and diseased individuals could be detected. Furthermore, the analysis revealed individual isotype (IgM, IgG, IgA)-specific α-TACA-AAbs profiles for the tested samples. MagPlex® beads that were coupled using the protocol for the KingFisherTM Flex yielded results that were comparable to those obtained with manually coupled beads.

Conclusion:
Taken together, we are now able to generate individual autoantibody profiles against glycan structures, using minor amounts of plasma, in a high throughput manner.

Background and aim:
Denmark is a world-wide leading country for the export of swine abroad: Annually 12 mio pigs (nucleus pigs, breeding sows, slaughter pigs and carcasses) are being exported to all continents of the world. The basis of this success is the fact that ALL Danish pig-farms cooperate in an organization (SEGES) that takes care of the health monitoring of the pigs at the farms. At any given moment SEGES knows which disease is present or absent on each individual farm. If a country, e.g. Russia, needs breeding sows, say 8.000 heads, of a certain health status, the SEGES organization knows exactly from which farms the breeding sows can be collected. Next days the pigs are collected and put on transport to Russia. This system, the Danish SPF (Specific Pathogen Free) system, is supported by ELISA-testing tot asses the status for the various diseases. However, the export is growing double digit annually and the testing increases to numbers which cannot be addressed in the classical way.

Methods:
Biovet started an evaluation project back in 2016/2017 in order to put all relevant ELISA tests on a MAGPIX® multiplexed system and …….

Results:
…… after ten-thousands of equivalence tests it became clear that the desired specificity and sensitivity of the multiplexed tests was even better than the individual ELISA tests. The SEGES organization implemented
the system in their testing lab, and 3 MAGPIX® machines operate daily to get the ever growing numbers of blood-samples tested, with good success. Each year one extra test is added to the multiplex.

Conclusion:
This project shows that very classical ELISA tests easily can be converted to multiplexed tests with enhanced test-characteristics. To such an extent that increases of numbers of tests can easily be run in the same easy setup of single ELISA’s. Currently, Biovet is expanding its international network of labs that can support health monitoring of pigs in general, in order to offer the same good level of testing everywhere were increasing numbers of animals are needed to feed the growing world-population.

Background:
The NMI applies miniaturized multiplex immunoassays for more than two decades. At the NMI commercially available multiplex immunoassays are applied for sample screening as well as newly developed custom specific assays. Over the last years a standard validation procedures was established which is routinely applied to our “home brew” as well as to commercially available multiplex immunoassays.

Methods:
Multiplex bead-based immunoassays on the Luminex platform were developed and thoroughly validated based on bioanalytical guidelines (EMA, FDA). To guarantee long term reproducibility and validity of assay performance a quality control system based on the Westgard Rules and Levey-Jennings-Plots was implemented.

Results:
Established assay development and validation procedures allow us a robust assessment of the capabilities of an assay and the evaluation of lot-to-lot variances during long term screening periods.

Conclusion:
In the time of extensive discussions on the lack of repeatability of scientific results, validation of reagents and assays, no matter whether they are commercially available or newly generated “home brew” assay, is a mandatory step forward to address such issues and allows to identify  strengths’ and weaknesses of the applied assay reagents during any sample screening process.

Purpose:
Molecular characterization of antimicrobial resistance determinants is recommended to monitor the mechanisms underlying antimicrobial resistance in indicator bacteria such as Escherichia coli. An affordable all-in-one alternative to whole genome sequencing is welcome to conduct the monitoring of critical antimicrobial resistance determinants in large collections of indicator or pathogenic bacteria in a one-health context

Methods:
Here we present a rapid genetic screening system based on a home-made multiplex Ligase Chain Reaction (LCR) assay using padlock probes coupled with a MagPlex-TAG beads hybridization and analysis on a Luminex® 200 TM platform. This 55-plex array, called the AMR-ARRAY, has been developed to detect the main antibiotic resistance determinants against β-lactams, (fluoro)quinolones, colistin, aminoglycosides and macrolides in E. coli, Shigella and Salmonella spp and can be used for other Enterobacteriaceae.

Results:
The mix of 55 probes was validated with available reference strains carrying the corresponding resistance gene or mutation. The array performed successfully on both reference and field strains. Four hundred eighty-one strains resistant to β-lactams, (fluoro)quinolones and/or colistin isolated in Belgium from food-producing animals (179 E. coli), food chain (117 E. coli), human samples (66 Salmonella spp. and 100 Shigella spp.) and 19 EURL-AR reference strains (9 E. coli and 10 Salmonella spp.) were assayed with a reduced version of this array (41-plex targeting β-lactams, (fluoro)quinolones and colistin resistance markers). Results were compared with phenotypic resistance profiles determined for the animal strains and with sequence-inferred resistance profiles determined for the other strains (using Sanger sequencing or PCR as reference method). The array performed well in detecting critical genetic determinants underlying antimicrobial resistance since 97.8% (175/179) of the food-producing animal strains yielded bead array profiles fully compatible with phenotypic resistance. For the genotype comparison, the array has a good performance too. The bead array profiles matched Sanger or PCR genotypes for 95.7% (112/117) of the food chain isolates, 97.6% (162/166) of the human isolates and 94.7% (18/19) of the inter-laboratory tests strains, giving a global genotypic concordance of 96.7% (292/302).

Conclusion:
We have developed in-house a 55-plex LCR/Luminex® MagPlex-TAG bead array able to detect the main antibiotic resistance determinants against β-lactams, (fluoro)quinolones, colistin, aminoglycosides and macrolides in Gram-negative bacteria. The performance of a reduced version of this array (41-plex targeting β-lactams, (fluoro)quinolones and colistin resistance markers) was evaluated with a collection of 481 field strains and a global concordance of 97.1% (467/481) was observed between the bead-array results and results obtained either by susceptibility testing or by genotyping using Sanger sequencing or PCR. The evaluation of the performance of the 55-plex AMR-ARRAY with field strains resistant to aminoglycosides and macrolides is ongoing.

Translational medicine has been fairly unsuccessful in translating innovation from the lab towards the clinics and back again. Many reasons account for the failure leading to a massive waste in life science research and the steep rise in costs of drug development and a failing pipeline from academia towards industry. The development of biomarkers suffers from similar problems. Despite all high hopes of advances such gene sequencing and multiplex technologies, translation from ‘big data’ into tangible therapies has been fairly unsuccessful with a few notable exceptions. Therefore, in order to truly advance translational medicine we need to examine carefully the root causes of the current problems and set out a completely new course, based on a paradigm shift in thinking about the way we collaborate and perform translational medicine.

Purpose:
Group B Streptococcus (GBS) is a Gram positive coccus which is part of the normal microbiota of the GI and GU tracts. However GBS is also a major cause of septicaemia and meningitis in newborns. Colonisation with GBS during pregnancy is a major risk factor for neonatal infection. Intrapartum antibiotic prophylaxis has greatly reduced incidence of early onset disease. However concerns over emerging antibiotic resistance and its impact on neonatal intestinal microbiota and a lack of protection afforded by intrapartum antibiotics outside of the first few days of life have led to GBS becoming a candidate for maternal vaccine development. Maternal IgG antibodies targeting capsular polysaccharides have been observed to provide protection against disease (Fabbrini et al 2016). Establishing a serocorrelate of protection could assist in the pathway to licensure of a GBS vaccine.

Methods:
A Bill and Melinda Gates Foundation funded consortium led by Kirsty Le Doare to establish a standardised multiplex immunoassay (MIA), based up luminex’s Xmap technology, and an opsonophagocytic killing assay (OPkA). This will be followed by a inter laboratory study between multiple partners to establish assay parameters and reproducibility.

Results:
Standardised reagents (CPS-PLL conjugates, standards) are being developed and characterised and preparations are being made for the inter laboratory study.

Conclusion:
Identifying a level of IgG which provides protection from GBS disease has been hampered by a lack of assay standardisation. A consortium has been established to develop standardised reagents and assays, including the multiplex immunoassay. Future studies to assess the relationship between antibody level, colonisation and disease are being planned.