GPCR/endocytosis/ERK signaling/S2R is involved in the regulation of the internalization, mitochondria-targeting and -activating properties of human salivary histatin 1.

Human salivary histatin 1 (Hst1) exhibits a series of cell-activating properties, such as promoting cell spreading, migration, and metabolic activity. We recently have shown that fluorescently labeled Hst1 (F-Hst1) targets and activates mitochondria, presenting an important molecular mechanism. However, its regulating signaling pathways remain to be elucidated. We investigated the influence of specific inhibitors of G protein-coupled receptors (GPCR), endocytosis pathways, extracellular signal-regulated kinases 1/2 (ERK1/2) signaling, p38 signaling, mitochondrial respiration and Na+/K+-ATPase activity on the uptake, mitochondria-targeting and -activating properties of F-Hst1. We performed a siRNA knockdown (KD) to assess the effect of Sigma-2 receptor (S2R) /Transmembrane Protein 97 (TMEM97)-a recently identified target protein of Hst1. We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1. Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1. The inhibitors of GPCR, ERK1/2, phagocytosis, and clathrin-mediated endocytosis (CME) as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake, which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity. Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1. We further showed the intracellular trafficking and targeting process of F-Hst1, in which early endosome plays an important role. Overall, phagocytosis, CME, GPCR, ERK signaling, and S2R/TMEM97 are involved in the internalization of Hst1, while only CME and S2R/TMEM97 are critical for its subcellular targeting. The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.

Natural and Synthetic Sortase A Substrates Are Processed by Staphylococcus aureus via Different Pathways.

Endogenous Staphylococcus aureus sortase A (SrtA) covalently incorporates cell wall anchored proteins equipped with a SrtA recognition motif (LPXTG) via a lipid II-dependent pathway into the staphylococcal peptidoglycan layer. Previously, we found that the endogenous S. aureus SrtA is able to recognize and process a variety of exogenously added synthetic SrtA substrates, including K(FITC)LPMTG-amide and K(FITC)-K-vancomycin-LPMTG-amide. These synthetic substrates are covalently incorporated into the bacterial peptidoglycan (PG) of S. aureus with varying efficiencies. In this study, we examined if native and synthetic substrates are processed by SrtA via the same pathway. Therefore, the effect of the lipid II inhibiting antibiotic bacitracin on the incorporation of native and synthetic SrtA substrates was assessed. Treatment of S. aureus with bacitracin resulted in a decreased incorporation of protein A in the bacterial cell wall, whereas incorporation of exogenous synthetic substrates was increased. These results suggest that natural and exogenous synthetic substrates are processed by S. aureus via different pathways.

Comparing periodontitis biomarkers in saliva, oral rinse and gingival crevicular fluid: A pilot study.

AIM: To explore the feasibility of screening for periodontitis by measuring biomarkers, namely total proteolytic activity (TPA), matrix metalloproteinase (MMP)-8, chitinase, lysozyme or their combination, in saliva, oral rinse and gingival crevicular fluid (GCF). MATERIAL AND METHODS: Subjects were recruited among healthy/gingivitis individuals and untreated periodontitis patients in Academic Centre for Dentistry Amsterdam (ACTA). All participants donated samples of unstimulated whole saliva, oral rinse and GCF. The protein concentrations and MMP-8 levels were determined by ELISA. Enzymatic activities were measured using appropriate fluorogenic substrates. RESULTS: In oral rinse samples, periodontitis patients (n = 19) exhibited significantly higher concentrations of MMP-8 and TPA than controls (n = 20). MMP-8 in combination with chitinase explained 88% of the variance and assigned a subject to control or periodontitis group, with best accuracy (87.2%) in oral rinse. CONCLUSIONS: The combination of MMP-8 and chitinase in the current oral rinse procedure has the potential to discriminate periodontitis from periodontal health/gingivitis.

Human Salivary Histatin-1 Promotes Osteogenic Cell Spreading on Both Bio-Inert Substrates and Titanium SLA Surfaces.

Promoting cell spreading is crucial to enhance bone healing and implant osteointegration. In this study, we investigated the stimulatory effect of human salivary histatin-1 (Hst-1) on the spreading of osteogenic cells in vitro as well as the potential signaling pathways involved. Osteogenic cells were seeded on bio-inert glass slides with or without the presence of Hst1 in dose-dependent or time-course assays. 1 scrambled and 6 truncated Hst1 variants were also evaluated. Cell spreading was analyzed using a well-established point-counting method. Fluorescent microscopy was adopted to examine the cellular uptake of fluorescently labeled Hst1 (F-Hst1) and also the cell spreading on sandblasted and acid etched titanium surfaces. Signaling inhibitors, such as U0126, SB203580, and pertussis toxin (PTx) were used to identify the potential role of extracellular-signal-regulated kinase, p38 and G protein-coupled receptor pathways, respectively. After 60 min incubation, Hst1 significantly promoted the spreading of osteogenic cells with an optimal concentration of 10 µM, while truncated and scrambled Hst1 did not. F-Hst1 was taken up and localized in the vicinity of the nuclei. U0126 and SB2030580, but not PTx, inhibited the effect of Hst1. 10 µM Hst1 significantly promoted the spreading of osteogenic cells on both bio-inert substrates and titanium SLA surfaces, which involved ERK and p38 signaling. Human salivary histatin-1 might be a promising peptide to enhance bone healing and implant osteointegration in clinic.

Salivary Histatin 1 and 2 Are Targeted to Mitochondria and Endoplasmic Reticulum in Human Cells.

Human salivary histatin 1 (Hst1) and Hst2 exhibit a series of cell-activating properties (e.g., promoting adhesion, spreading, migration and metabolic activity of mammalian cells). In contrast, Hst5 shows an anti-fungal property but no cell-activating properties. Previous findings suggest that their uptake and association with subcellular targets may play a determinant role in their functions. In this study, we studied the uptake dynamics and subcellular targets of Hst1, Hst2 and Hst5 in epithelial cells (HO1N1 human buccal carcinoma epithelial cell line). Confocal laser scanning microscopy (CLSM) revealed that fluorescently labeled Hst1 (F-Hst1) was taken up into the intracellular space of epithelial cells. Then, 60 min post-incubation, the total fluorescence of cell-associated F-Hst1, as measured using flow cytometry, was significantly higher compared to those of F-Hst2 and F-Hst5. In contrast, virtually no association occurred using the negative control-scrambled F-Hst1 (F-Hstscr). CLSM images revealed that F-Hst1, 2 and 5 co-localized with mitotrackerTM-labeled mitochondria. In addition, F-Hst1 and F-Hst2 but neither F-Hst5 nor F-Hst1scr co-localized with the ER-trackerTM-labeled endoplasmic reticulum. No co-localization of Hst1, 2 and 5 with lysosomes or the Golgi apparatus was observed. Furthermore, Hst1 and Hst2 but not Hst5 or Hst1scr significantly promoted the metabolic activity of both human epithelial cell lines, HaCaT human keratinocytes and primary human gingival fibroblasts.

All-trans retinoic acid and human salivary histatin-1 promote the spreading and osteogenic activities of pre-osteoblasts in vitro.

Cell-based bone tissue engineering techniques utilize both osteogenic cells and biomedical materials, and have emerged as a promising approach for large-volume bone repair. The success of such techniques is highly dependent on cell adhesion, spreading, and osteogenic activities. In this study, we investigated the effect of co-administration of all-trans retinoic acid (ATRA) and human salivary peptide histatin-1 (Hst1) on the spreading and osteogenic activities of pre-osteoblasts on bio-inert glass surfaces. Pre-osteoblasts (MC3T3-E1 cell line) were seeded onto bio-inert glass slides in the presence and absence of ATRA and Hst1. Cell spreading was scored by measuring surface areas of cellular filopodia and lamellipodia using a point-counting method. The distribution of fluorogenic Hst1 within osteogenic cells was also analyzed. Furthermore, specific inhibitors of retinoic acid receptors α, ß, and γ, such as ER-50891, LE-135, and MM-11253, were added to identify the involvement of these receptors. Cell metabolic activity, DNA content, and alkaline phosphatase (ALP) activity were assessed to monitor their effects on osteogenic activities. Short-term (2 h) co-administration of 10 µm ATRA and Hst1 to pre-osteoblasts resulted in significantly higher spreading of pre-osteoblasts compared to ATRA or Hst1 alone. ER-50891 and LE-135 both nullified these effects of ATRA. Co-administration of ATRA and Hst1 was associated with significantly higher metabolic activity, DNA content, and ALP activity than either ATRA or Hst1 alone. In conclusion, co-administration of Hst1 with ATRA additively stimulated the spreading and osteogenicity of pre-osteoblasts on bio-inert glass surfaces in vitro.

Please try harder! The influence of hearing status and evaluative feedback during listening on the pupil dilation response, saliva-cortisol and saliva alpha-amylase levels.

The pupil dilation response is sensitive both to listening effort and the emotional significance of a task. We aimed to assess the influence of evaluative feedback on the pupil dilation response using a speech reception threshold (SRT) task. Besides the pupil dilation response, we acquired subjective ratings and two physiological biomarkers sensitive to stress: cortisol and alpha-amylase levels as determined in saliva samples. We included 34 participants with normal hearing (mean age = 52 years, age range 25-67 years) and 29 age-matched participants with mild-to-moderate hearing loss (mean age = 52 years, age range 23-64 years). Half of the participants performed a standard SRT test without feedback, and the other half performed an SRT test in which they did receive feedback and were urged to perform better. The SRT conditions targeted 50% and 71% correct reception of the sentences. Pupil size was recorded and saliva samples were obtained and participants rated their experience of the task. Participants with hearing loss performed more poorly on the SRT test than participants with normal hearing participants receiving feedback had better SRTs in the 71% intelligibility condition and higher peak pupil dilation in both intelligibility conditions than participants who did not receive feedback, irrespective of hearing status. Saliva cortisol level and alpha-amylase activity reflected the usual diurnal patterns but showed no effects of hearing status or feedback. Finally, participants who received feedback experienced more difficulties than those who did not receive feedback, irrespective of hearing status. This study underlines the importance of taking into account the influence of task instructions and feedback in a speech perception task as these factors may influence the experienced difficulties, listening effort, and task performance.

LFchimera protects HeLa cells from invasion by Yersinia spp. in vitro.

Yersinia pestis is the causative agent of plague. As adequate antibiotic treatment falls short and currently no effective vaccine is available, alternative therapeutic strategies are needed. In order to contribute to solving this problem we investigated the therapeutic potential of the peptide construct LFchimera against the safer-to-handle Y. pestis simulants Yersinia enterocolitica and Yersinia pseudotuberculosis in vitro. LFchimera is a heterodimeric peptide construct mimicking two antimicrobial domains of bovine lactoferrin, i.e. lactoferrampin and lactoferricin. LFchimera has been shown to be a potent antimicrobial peptide against a variety of bacteria in vitro and in vivo. Also Y. enterocolitica and Y. pseudotuberculosis have been shown to be susceptible for LFchimera in vitro. As Yersiniae spp. adhere to and invade host cells upon infection, we here investigated the effects of LFchimera on these processes. It was found that LFchimera has the capacity to inhibit host-cell invasion by Yersiniae spp. in vitro. This effect appeared to be host-cell mediated, not bacteria-mediated. Furthermore it was found that exposure of human HeLa epithelial cells to both LFchimera and the bacterial strains evoked a pro-inflammatory cytokine release from the cells in vitro.

Salivary peptide histatin 1 mediated cell adhesion: a possible role in mesenchymal-epithelial transition and in pathologies.

Histatins are histidine-rich peptides present in the saliva of humans and higher primates and have been implicated in the protection of the oral cavity. Histatin 1 is one of the most abundant histatins and recent reports show that it has a stimulating effect on cellular adherence, thereby suggesting a role in maintaining the quality of the epithelial barrier and stimulating mesenchymal-to-epithelial transition. Here we summarize these findings and discuss them in the context of previous reports. The recent findings also provide new insights in the physiological functions of histatin 1, which are discussed here. Furthermore, we put forward a possible role of histatin 1 in various pathologies and its potential function in clinical applications.

Anthrax protective antigen is a calcium-dependent serine protease.

Bacillus anthracis secretes a three component exotoxin-complex, which contributes to anthrax pathogenesis. Formation of this complex starts with the binding of protective antigen (PA) to its cellular receptor. In this study, we report that PA is a calcium-dependent serine protease and that the protein potentially uses this proteolytic activity for receptor binding. Additionally our findings shed new light on previous research describing the inhibition of anthrax toxins and exotoxin formation. Importantly, inhibition of the proteolytic activity of protective antigen could be a novel therapeutic strategy in fighting B. anthracis-related infections.

Polymorphonuclear neutrophil integrity and functionality are preserved when exposed to saliva.

OBJECTIVE: Polymorphonuclear neutrophils (PMNs) are the most abundant innate immune cells and are also important effectors in the maintenance of oral health. However, little is known about the effects of saliva on the PMN. We therefore aimed to investigate the effect of saliva on the PMNs' morphology and functioning. DESIGN: Effect of saliva on the membrane integrity of PMNs isolated from blood was evaluated with FACS using Annexin V (apoptosis marker) and propidum iodide (membrane integrity marker). The effect on cell morphology was examined using transmission electron imaging. Binding and phagocytosis of the oral bacterium Fusobacterium nucleatum by PMNs was analysed by FACS. Reactive oxygen species (ROS) production was measured with chemiluminescence. RESULTS: Incubation with saliva for 60 min had no detectable effects on the membrane integrity or the morphology of PMNs. In contrast, preincubation of F. nucleatum with saliva inhibited its subsequent interaction with PMNs, resulting in a diminished production of ROS. CONCLUSIONS: Saliva does not impair the function of PMNs. However, interaction of salivary components with F. nucleatum may affect their recognition by PMNs resulting in a diminished functional response.

High number of chromosomal copy number aberrations inversely relates to t(11;19)(q21;p13) translocation status in mucoepidermoid carcinoma of the salivary glands.

Although rare, mucoepidermoid carcinoma (MEC) is one of the most common malignant salivary gland tumors. The presence of the t(11;19)(q21;p13) translocation in a subset of MECs has raised interest in genomic aberrations in MEC. In the present study we conducted genome-wide copy-number-aberration analysis by micro-array comparative-genomic-hybridization on 27 MEC samples. Low/intermediate-grade MECs had significantly fewer copy-number-aberrations compared to high-grade MECs (low vs high: 3.48 vs 30; p = 0.0025; intermediate vs high: 5.7 vs 34.5; p = 0.036). The translocation-negative MECs contained more copy-number-aberrations than translocation-positive MECs (average amount of aberrations 15.9 vs 2.41; p =0.04). Within all 27 MEC samples, 16p11.2 and several regions on 8q were the most frequently gained regions , while 1q23.3 was the most frequently detected loss. Low/intermediate-grade MEC samples had copy-number-aberrations in chromosomes 1, 12 and 16, while high-grade MECs had a copy-number-aberration in 8p. The most commonly observed copy-number-aberration was the deletion of 3p14.1, which was observed in 4 of the translocation-negative MEC samples. No recurrent copy-number-aberrations were found in translocation-positive MEC samples. Based on these results, we conclude that MECs may be classified as follows: (i) t(11;19)(q21;p13) translocation-positive tumors with no or few chromosomal aberrations and (ii) translocation-negative tumors with multiple chromosomal aberrations.

Saliva-Derived Host Defense Peptides Histatin1 and LL-37 Increase Secretion of Antimicrobial Skin and Oral Mucosa Chemokine CCL20 in an IL-1α-Independent Manner.

Even though skin and oral mucosae are continuously in contact with commensal and opportunistic microorganisms, they generally remain healthy and uninflamed. Host defense peptides (HDPs) make up the body's first line of defense against many invading pathogens and are involved in the orchestration of innate immunity and the inflammatory response. In this study, we investigated the effect of two salivary HDPs, LL-37 and Hst1, on the inflammatory and antimicrobial response by skin and oral mucosa (gingiva) keratinocytes and fibroblasts. The potent antimicrobial chemokine CCL20 was investigated and compared with chemokines CCL2, CXCL1, CXCL8, and CCL27 and proinflammatory cytokines IL-1α and IL-6. Keratinocyte-fibroblast cocultures showed a synergistic increase in CCL20 secretion upon Hst1 and LL-37 exposure compared to monocultures. These cocultures also showed increased IL-6, CXCL1, CXCL8, and CCL2 secretion, which was IL-1α dependent. Secretion of the antimicrobial chemokine CCL20 was clearly IL-1α independent. These results indicate that salivary peptides can stimulate skin as well as gingiva cells to secrete antimicrobial chemokines as part of the hosts' defense to counteract infection.

Mucoepidermoid carcinoma-associated expression of MUC5AC, MUC5B and mucin-type carbohydrate antigen sialyl-Tn in the parotid gland.

OBJECTIVES: The aberrant expression of mucins and mucin-type carbohydrates has been described in many types of cancer, including mucoepidermoid carcinoma (MEC), a malignant salivary gland tumor. In this study, we examined the aberrant expression patterns of mucins (MUC1, MUC4, MUC5AC and MUC5B), simple mucin-type carbohydrate antigens (Tn, sialyl-Tn and T) and mature carbohydrate antigens (Lewisa and sulfo-Lewisa antigens) in MEC originating from the parotid gland, which normally does not secrete mucins. DESIGN: We conducted an immunohistochemical study to investigate the presence of mucins and carbohydrates in 24 MEC samples originating from the parotid gland and in surrounding normal tissue of the same gland in comparison 6 samples of normal salivary glands. The expression levels were compared with respect to the histological grading. Furthermore, 24 MEC samples from non-parotid salivary glands were included. RESULTS: We observed loss of topology of membrane-bound MUC1 and MUC4, and de novo expression of MUC5AC, MUC5B and sialyl-Tn in MEC that originated in the parotid gland. Furthermore, mucins MUC1, MUC4 and carbohydrate antigens Tn, sialyl-Tn, T, Lewisa and sulfo-Lewisa were overexpressed in MEC samples compared to surrounding normal salivary gland tissues. MUC1 was expressed in both low- and high grade MECs, whereas MUC4 was not expressed in high grade MECs of the parotid gland. CONCLUSION: During the development of MEC in the parotid gland, the genes for gel-forming secretory mucins are switched on. Besides these MEC tissues overexpress short oligosaccharides, suggesting that the glycosylation machinery is altered.

Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Histatins are multifunctional histidine-rich peptides secreted by the salivary glands and exclusively present in the saliva of higher primates, where they play a fundamental role in the protection of the oral cavity. Our previously published results demonstrated that histatin-1 (Hst1) promotes cell-substrate adhesion in various cell types and hinted that it could also be involved in cell-cell adhesion, a process of fundamental importance to epithelial and endothelial barriers. Here we explore the effects of Hst1 on cellular barrier function. We show that Hst1 improved endothelial barrier integrity, decreased its permeability for large molecules, and prevented translocation of bacteria across epithelial cell layers. These effects are mediated by the adherens junction protein E-cadherin (E-cad) and by the tight junction protein zonula occludens 1, as Hst1 increases the levels of zonula occludens 1 and of active E-cad. Hst1 may also promote epithelial differentiation as Hst1 induced transcription of the epithelial cell differentiation marker apolipoprotein A-IV (a downstream E-cad target). In addition, Hst1 counteracted the effects of epithelial-mesenchymal transition inducers on the outgrowth of oral cancer cell spheroids, suggesting that Hst1 affects processes that are implicated in cancer progression.-Van Dijk, I. A., Ferrando, M. L., van der Wijk, A.-E., Hoebe, R. A., Nazmi, K., de Jonge, W. J., Krawczyk, P. M., Bolscher, J. G. M., Veerman, E. C. I., Stap, J. Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

The scavenging capacity of DMBT1 is impaired by germline deletions.

The Scavenger Receptor Cysteine-Rich (SRCR) proteins are an archaic group of proteins characterized by the presence of multiple SRCR domains. They are membrane-bound or secreted proteins, which are generally related to host defense systems in animals. Deleted in Malignant Brain Tumors 1 (DMBT1) is a SRCR protein which is secreted in mucosal fluids and involved in host defense by pathogen binding by its SRCR domains. Genetic polymorphism within DMBT1 leads to DMBT1-alleles giving rise to polypeptides with interindividually different numbers of SRCR domains, ranging from 8 SRCR domains (encoded by 6 kb DMBT1 variant) to 13 SRCR domains (encoded by the 8 kb DMBT1 variant). In the present study, we have investigated whether reduction from 13 to 8 amino-terminal SRCR domains leads to reduction of bacterial binding. The 6 kb variant bound ~20-45% less bacteria compared to the 8 kb variant. These results support the hypothesis that genetic variation in DMBT1 may influence microbial defense.

Effects of lactoferrin derived peptides on simulants of biological warfare agents.

Lactoferrin (LF) is an important immune protein in neutrophils and secretory fluids of mammals. Bovine LF (bLF) harbours two antimicrobial stretches, lactoferricin and lactoferampin, situated in close proximity in the N1 domain. To mimic these antimicrobial domain parts a chimeric peptide (LFchimera) has been constructed comprising parts of both stretches (LFcin17-30 and LFampin265-284). To investigate the potency of this construct to combat a set of Gram positive and Gram negative bacteria which are regarded as simulants for biological warfare agents, the effect on bacterial killing, membrane permeability and membrane polarity were determined in comparison to the constituent peptides and the native bLF. Furthermore we aimed to increase the antimicrobial potency of the bLF derived peptides by cationic amino acid substitutions. Overall, the bactericidal activity of the peptides could be related to membrane disturbing effects, i.e. membrane permeabilization and depolarization. Those effects were most prominent for the LFchimera. Arginine residues were found to be crucial for displaying antimicrobial activity, as lysine to arginine substitutions resulted in an increased antimicrobial activity, affecting mostly LFampin265-284 whereas arginine to lysine substitutions resulted in a decreased bactericidal activity, predominantly in case of LFcin17-30.

The Effect of Exercise on Salivary Viscosity.

A common experience after exercise is the presence of a thick and sticky saliva layer on the oral surfaces, which causes a feeling of a dry mouth. Since the salivary mucin MUC5B is responsible for the visco-elastic behavior of saliva, in the present study we explored the effect of exercise on both the salivary viscosity and the secretion of MUC5B in saliva. Twenty healthy dental students performed an aerobic exercise by cycling for 15 min on cycle-ergometers at a heart rate of 130-140 beats per minute. Saliva was collected at three time points: before exercise, immediately after exercise and after 30 min recovery. Salivary flow rate, viscosity, amylase activity, total protein, carbohydrate and MUC5B concentration were determined. Salivary flow rate, protein and amylase did not change significantly. Immediately after exercise, the salivary viscosity and carbohydrate concentration were significantly higher than at baseline and after 30 min recovery. Immediately after exercise, the MUC5B concentration was significantly higher than after 30 min recovery. It is concluded that the presence of thick saliva after exercise is at least partially due to an increased secretion of MUC5B.

Incorporation of a Valine-Leucine-Lysine-Containing Substrate in the Bacterial Cell Wall.

The emergence of antibiotic-resistant bacteria is a major public health threat, and therefore novel antimicrobial targets and strategies are urgently needed. In this regard, cell-wall-associated proteases are envisaged as interesting antimicrobial targets due to their role in cell wall remodeling. Here, we describe the discovery and characteristics of a protease substrate that is processed by a bacterial cell-wall-associated protease. Stationary-phase grown Gram-positive bacteria were incubated with fluorogenic protease substrates, and their cleavage and covalent incorporation into the cell wall was analyzed. Of all of the substrates used, only one substrate, containing a valine-leucine-lysine (VLK) motif, was covalently incorporated into the bacterial cell wall. Linkage of the VLK-peptide substrate appeared unrelated to sortase A and B activity, as both wild-type and sortase A and B knock out Staphylococcus aureus strains incorporated this substrate into their cell wall with comparable efficiency. Additionally, the VLK-peptide substrate showed significantly higher incorporation in the cell wall of VanA-positive Enterococcus faecium strains than in VanB- and vancomycin-susceptible isolates. In conclusion, the VLK-peptide substrate identified in this study shows promise as a vehicle for targeting antimicrobial compounds and diagnostic contrast agents to the bacterial cell wall.

Comparison of non-magnetic and magnetic beads in bead-based assays.

Multiplex bead-based flow cytometry is an attractive way for simultaneous, rapid and cost-effective analysis of multiple analytes in a single sample. Previously, we developed various bead-based assays using non-magnetic beads coated with Staphylococcus aureus and Streptococcus pneumoniae antigens for the detection of antibodies. Here, we compared the performance of the assay using non-magnetic beads with one based on the newly developed magnetic beads. We optimized the magnetic beads' coupling procedure and antibody detection assays for S. aureus and S. pneumoniae antigens and we measured IgG in human pooled serum against a series of S. aureus and S. pneumoniae-derived antigens in a singleplex and in a multiplex assay, respectively. For the multiplex assay, the comparison between magnetic and non-magnetic beads showed: i) in the majority of the cases (13 of the 17 tested S. pneumoniae antigens) significantly higher Median Fluorescence Intensity (MFI) values, ii) lower detection limits, iii) lower coefficient of variation (CV: 12% vs. 7% for non-magnetic vs. magnetic beads), so lower inter-assay variation and hence higher reproducibility. Magnetic bead coupling is cost effective, as we used 25% of the normal amount of antigen and only 50% of the beads in comparison to the non-magnetic beads. This optimized magnetic-based assay, which combines ease of use with an improved assay performance, allows detection of antibodies with a low titer that are potentially missed with the non-magnetic-based assay.