Plasma-Activated Tap Water with Oxidative Potential Has an Inactivating Effect on Microbiological Contaminants in Aqueous Suspensions.

Plasma-activated water (PAW) generated from tap water has gained attention as a disinfectant when used directly in its pure form. Little is known about the application of PAW for bacterial inactivation in aqueous environments because its use in fluids results in dilutions. We investigated the effect of PAW in aqueous suspensions simulating such dilutions, and we focused on the minimal addition of PAW volumes to bacterial aqueous suspensions still resulting in high inactivation rates. The antimicrobial effect was highly dependent on the activation of PAW. An increase in activation power from 90 to 100 W resulted in a greater microbial reduction with an identical 10 min activation time. The susceptibility to PAW dilutions was analyzed in detail regarding nine Gram-negative species out of Enterobacterales and other waterborne microorganisms as well as four Gram-positive species present in two different matrices, in saline and in tap water, at high concentrations simulating massive contamination situations. For this purpose, the PAW activation setting of 90 W and 30 min was defined in order to be able to differentiate the limitations of inactivation in individual bacterial species. The Gram-negatives in saline demonstrated susceptibility when one volume unit of PAW was added. However, twice the PAW volume was necessary for inactivation when bacteria were present in tap water. Gram-positive microorganisms were more robust, indicated by prolonged contact times before inactivation. Our results indicate that PAW can be used for bacterial decontamination processes in aqueous environments when added in surplus. Optimized activation settings such as electric power to generate PAW and the contact times to the samples increase the effect of the inactivation a wide range of bacteria, regardless of their resistance profiles.

Sink Drains in a Neonatal Intensive Care Unit: A Retrospective Risk Assessment and Evaluation.

Water systems in health care facilities can form reservoirs for Gram-negative bacteria. While planning a new neonatal intensive care unit (NICU), we performed a retrospective evaluation of potential risks from water-diverting systems on the existing NICU of our tertiary care University Hospital. During 2017 to 2023, we recorded nine nosocomial cluster events with bacterial pathogens in our NICU. Of these, three clusters of Gram-negative bacteria were potentially related to sink drains: A Klebsiella oxytoca, a Pseudomonas aeruginosa, and an Enterobacter hormaechei cluster were uncovered by clinical routine screening of patients and breastmilk samples. They were confirmed using whole-genome sequencing and a subsequent core genome multilocus sequence typing (cgMLST) algorithm. Our observations highlight that the implementation of sink drains in a NICU may have negative effects on patients' safety. Construction planning should concentrate on the avoidance of washbasins in patient rooms when redesigning sensitive areas such as NICUs.

Occurrence and prevalence of Legionella species in dental chair units in Germany with a focus on risk factors.

PURPOSE: Water-bearing instruments and treatments in dental units produce aerosols originating from the dental unit waterlines (DUWLs), which are often microbially contaminated. Particularly, the presence of Legionella mainly realized as aerosols leads to a risk of infection in patients and dental staff. METHODS: Here, we record the general bacteriological status of DUWLs in Germany and investigated the prevalence of Legionella spp., with a focus on identification and occurrence of distinct species considering the various aspects of dental practice such as dental chair equipment, disinfection methods, and temperatures. RESULTS: Out of 3789 water samples of 459 dental practices, collected in the years 2019 and 2020, 36.4% were Legionella positive with predominance of L. anisa (97.89%) identified by MALDI-TOF biotyping. L. pneumophila was detected very rarely. Risk factor analysis revealed that temperatures >20°C are a significant factor for increased Legionella colonization. CONCLUSION: In order to minimize the risk of infection, routine monitoring of the water quality in dental chair units is recommended with regard to general microbiological loads and to the presence of Legionella as opportunistic pathogen as well as the regular application of routine disinfection procedures.

Impact of Chlorine Dioxide on Pathogenic Waterborne Microorganisms Occurring in Dental Chair Units.

Bacterial contamination is a problem in dental unit water lines with the consequence of implementing regular disinfection. In this study, the short-term impact of chlorine dioxide (ClO2) treatment was investigated on the microorganisms Legionella pneumophila and L. anisa, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. The environmental background was proven as an important factor regarding the tolerance to 0.4 mg/L ClO2 as saline and phosphate-buffered saline resulted in a higher bacterial reduction than tap water. Gram-positive microorganisms demonstrated higher robustness to ClO2 than Gram-negative, and microorganisms adapted to tap water showed increased stability compared to cultured cells. At high densities, substantial numbers of bacteria were able to withstand disinfection, whereby the use of 4.6 mg/L ClO2 increased the inactivation rate. A massive cell decrease occurred within the first 5 minutes with subsequent plateau formation or slowed cell reduction upon further exposure. This biphasic kinetics cannot be explained by a ClO2 depletion effect alone, because the probability of bacterial subpopulations with increased tolerance should be taken into account, too. Our results prove high disinfection efficiency to microorganisms that were rather found in correlation to the level of bacterial contamination and background solutions than the chosen concentration for ClO2 treatment itself.

[Opinion Survey of Paediatricians on Vaccination Status of Refugee Children - Challenges of a Medical Connection to the Regular Outpatient Care System]. / Meinungsbild von Pädiatern zum Impfstatus von geflüchteten Kindern ­ Herausforderungen einer medizinischen Anbindung an das ambulante Regelversorgungssystem.

BACKGROUND: In addition to the primary health care of refugees, their integration into the regular outpatient care system should be ensured. Initial data suggest that a gap of vaccination among (school) children of refugee families might have emerged in the period between the first general inspection on arrival (the first central health measure) and the transition to the local health care system. OBJECTIVES: The aim of this study was to obtain the opinion of practicing paediatricians regarding the vaccination status of refugee children to examine whether a variance in the measles, mumps, rubella (varicella) vaccination schedule might have emerged between the periods of initial admission and school enrolment examination. Evaluations of both inhibiting and promoting conditions should generate fields of action regarding the systematic integration into the regular health care system. METHOD: Qualitative interviews with experts as well as a quantitative questionnaire survey to measure the opinion of registered paediatricians in Münster were analyzed. RESULTS: The assessments showed that there was no clear vaccination gap among (school) children of refugee families. One challenge was the systematic integration into the local outpatient care system. Critical issues were inadequate vaccination education, language barriers, and frequent changes in location. The vaccination engagement and vaccination behaviour of refugees were assessed as most positive. International standards, in particular the sphere standards, attracted insufficient attention in practical implementation within the refugee relief programs. CONCLUSIONS: Based on the results, it is possible to identify fields of action for the prevention of vaccination gaps among refugees as well as for their systematic integration into the regular outpatient care system. The sphere standards as international standards should be incorporated more consciously.

Development of a MALDI-TOF MS-based screening panel for accelerated differential detection of carbapenemases in Enterobacterales using the direct-on-target microdroplet growth assay.

Carbapenemase-producing bacteria are a growing issue worldwide. Most phenotypic detection methods are culture-based, requiring long incubation times. We present a phenotypic screening panel for detection of carbapenem non-susceptibility and differentiation of carbapenemase classes and AmpC, the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA). It was validated on 7 reference strains and 20 challenge Enterobacterales isolates. Broth microdilution (BMD) and combination disk test (CDT) were also performed, as well as PCR as reference method. The panel based on the synergy between meropenem and carbapenemase inhibitors, determined by incubating these substances with bacterial suspension on a MALDI-TOF MS target and subsequently assessing bacterial growth on the target's spots by MS. After 4 hours of incubation, DOT-MGA correctly identified KPC, MBL and OXA (100% agreement with PCR). Detection of AmpC coincided with BMD and CDT but agreement with PCR was low, not ruling out false negative PCR results. DOT-MGA delivered more accurate results than BMD and CDT in a significantly shorter time, allowing for detection of carbapenem non-susceptibility, MIC determination and carbapenemase differentiation in one step.

Prevalence of latent tuberculosis in homeless persons: A single-centre cross-sectional study, Germany.

PURPOSE: Homeless persons have a high risk for tuberculosis. The prevalence of latent tuberculosis infection and the risk for a progression to active tuberculosis is higher in the homeless than in the general population. The objective was to assess the prevalence and risk factors of tuberculosis/latent tuberculosis infection in a homeless population in Germany. METHODS: Homeless individuals (n = 150) were enrolled in a cross-sectional study at three shelters in Münster, Germany (October 2017-July 2018). All participants were screened using an ELISPOT interferon-γ release assay (IGRA). Those participants tested positive/borderline by IGRA provided three sputa for microbiological analysis (line probe assay, microscopy, culture) and underwent a chest X-ray to screen for active pulmonary TB. Risk factors for tuberculosis/latent tuberculosis infection were analysed using a standardized questionnaire. RESULTS: Of the 142 evaluable IGRA, 21 (15%) were positive and two (1%) were borderline. No participant with a positive/borderline IGRA had an active tuberculosis as assessed by chest X-ray and microbiology. A negative IGRA was associated with a citizenship of a low-incidence country for tuberculosis (according to WHO, p = 0.01), low-incidence country of birth (p<0.001) or main residence in a low-incidence country in the past five years (p = 0.002). CONCLUSIONS: The prevalence of latent tuberculosis infection (diagnosed by a positive/borderline IGRA) was 16%; no active tuberculosis was detected. The highest risk for latent tuberculosis infection was found in patients from high-incidence countries. This population at risk should be either treated for latent tuberculosis infection or need to be monitored to early detect a progression into active disease.

Effect of chlorine on cultivability of Shiga toxin producing Escherichia coli (STEC) and ß-lactamase genes carrying E. coli and Pseudomonas aeruginosa.

The worldwide spread of toxin-producing and multi-drug resistant bacteria in water, food and the environment is considered a major threat to human health. Drinking water quality is controlled by inspection of fecal indicators presence whereby viable contaminants will be efficiently reduced by chlorination which is a common process for disinfection. However, the all-out efficiency is arguable, because bacterial regrowth has been documented after disinfection. In this study, we investigated the stability of Shiga toxin producing Escherichia coli (STEC) and ß-lactamase expressing E. coli and Pseudomonas aeruginosa isolates, both equipped with multiple or single ß-lactamase resistance genes. The aim of the study was to analyze the efficiency of chlorine (Cl2) disinfection against shigatoxigenic or ß-lactamase producing bacteria. Cl2 reacts with the bacterial cells after first contact. Counts of antibiotic resistant E. coli were lower after short than upon extended Cl2 treatment. P. aeruginosa counts decreased moderately upon 15-60 min treatment with 1.2 mg Cl2/l, while cells adapted to tap water were not cultivable anymore. We assume that the bacterial physiology changed to a temporary non-cultivatable state at first Cl2 contact followed by resuscitation of some cells at later stages. STEC viability went down continuously at low Cl2 concentrations and these toxigenic E. coli isolates exhibited slightly increased stability to Cl2 treatment compared with non-toxigenic E. coli. Controlling the efficiency of disinfection, realistic counts of cultivatable cells are achieved after extended Cl2 action.

Distinct Expression of Immunoglobulin-Binding Proteins in Shiga Toxin-Producing Escherichia coli Implicates High Protein Stability and a Characteristic Phenotype.

Several immunoglobulin-binding proteins of Escherichia coli (Eib) have been isolated from both non-pathogenic and pathogenic E. coli strains. Shiga toxin (Stx)-producing E. coli (STEC) contain eibG either as a single gene or in combination with eibC, while other E. coli strains harbour single or multiple eib genes. The Eib proteins bind human immunoglobulins in a non-immune manner and contribute to bacterial chain-like adherence to human epithelial cells. In this study, the EibG expression in several STEC strains was analysed under different environmental conditions. STEC produced high levels of EibG in complex media and lower levels in low-grade and minimal media under static growth conditions. This characteristic was independent on the Eib subtypes. Microscopically, EibG-expressing STEC exhibited chain formation and aggregation in all employed media, while aggregates were only visible after growth in complex medium. Once expressed, EibG proteins demonstrate high stability during prolonged incubation. Our findings indicate that the regulation of the expression of Eib proteins is highly complex, although the protein levels vary among STEC strains. However, positive upregulation conditions generally result in distinct phenotypes of the isolates.

Bacterial contamination of water samples in Gabon, 2013.

Contamination of water is a major burden in the public health setting of developing countries. We therefore assessed the quality of water samples in Gabon in 2013. The main findings were a contamination rate with coliforms of 13.5% and the detection of a possible environmental reservoir for extended spectrum beta-lactamase-producing bacteria.

Genetic, histochemical and biochemical studies on goat TSE cases from Cyprus.

Scrapie and bovine spongiform encephalopathy (BSE) are transmissible spongiform encephalopathies (TSE's) affecting sheep and goats. Susceptibility of goats to scrapie is influenced by polymorphisms of the prion protein gene (PRNP) of the host. Five polymorphisms are associated with reduced susceptibility to TSE's. In the study presented here caprine samples from a scrapie eradication program on Cyprus were genotyped and further characterized using BioRad TeSeE rapid test, histological, immunohistochemical and biochemical methods. In total 42 goats from 20 flocks were necropsied from which 25 goats showed a positive result in the rapid test, a spongiform encephalopathy and an accumulation of pathological prion protein (PrPSc) in the obex. PrPSc deposits were demonstrated in the placenta, peripheral nervous and lymphoreticular system. Two animals showed PrPSc-accumulations in peripheral tissues only. By discriminatory immunoblots a scrapie infection could be confirmed for all cases. Nevertheless, slight deviations in the glycosylation pattern might indicate the presence of different scrapie strains. Furthermore scrapie samples from goats in the current study demonstrated less long term resistance to proteinase K than ovine or caprine BSE control samples. Reduced scrapie susceptibility according to the PRNP genotype was demonstrated (Fishers Exact test, p < 0.05) for the goats with at least one polymorphism (p = 0.023) at the six codons examined and in particular for those with polymorphisms at codon 146 (p = 0.016). This work characterizes scrapie in goats having implications for breeding and surveillance strategies.

Copper and Zinc Interactions with Cellular Prion Proteins Change Solubility of Full-Length Glycosylated Isoforms and Induce the Occurrence of Heterogeneous Phenotypes.

Prion diseases are characterized biochemically by protein aggregation of infectious prion isoforms (PrPSc), which result from the conformational conversion of physiological prion proteins (PrPC). PrPC are variable post-translationally modified glycoproteins, which exist as full length and as aminoterminally truncated glycosylated proteins and which exhibit differential detergent solubility. This implicates the presence of heterogeneous phenotypes, which overlap as protein complexes at the same molecular masses. Although the biological function of PrPC is still enigmatic, evidence reveals that PrPC exhibits metal-binding properties, which result in structural changes and decreased solubility. In this study, we analyzed the yield of PrPC metal binding affiliated with low solubility and changes in protein banding patterns. By implementing a high-speed centrifugation step, the interaction of zinc ions with PrPC was shown to generate large quantities of proteins with low solubility, consisting mainly of full-length glycosylated PrPC; whereas unglycosylated PrPC remained in the supernatants as well as truncated glycosylated proteins which lack of octarepeat sequence necessary for metal binding. This effect was considerably lower when PrPC interacted with copper ions; the presence of other metals tested exhibited no effect under these conditions. The binding of zinc and copper to PrPC demonstrated differentially soluble protein yields within distinct PrPC subtypes. PrPC-Zn2+-interaction may provide a means to differentiate glycosylated and unglycosylated subtypes and offers detailed analysis of metal-bound and metal-free protein conversion assays.

Population structure of Legionella spp. from environmental samples in Gabon, 2013.

Aquatic environments are the most important source for Legionella spp. infections such as Legionnaires' disease and Pontiac fever. The reservoirs of Legionella spp. are mostly unclear in sub-Saharan Africa. The aim of this study, conducted in 2013, was to identify geographical areas of an increased risk for exposure to Legionella spp., and to describe the population structure of Legionella spp. from different water sources in a cross-sectional study in Gabon. Fresh water samples (n = 200) were cultured on Legionella selective agar; species were confirmed by MALDI-TOF, a Legionella pneumophila specific real-time PCR and 16S RNA gene sequencing. Serogroups were identified by agglutination test. The population structure was assessed by multilocus sequence typing (MLST). Legionella spp. isolates (n = 29) were frequently found in the hospital setting particularly in hot water systems. Open water bodies (i.e. rivers, lakes) were not contaminated with Legionella spp. Isolated L. pneumophila mainly belonged to serogroups 2-14 (n = 19) and MLST sequence type ST1, ST75 (and related STs) and ST1911. In conclusion, hospitalized patients might have an increased risk to become infected with Legionella spp. in the studied areas in Gabon, particularly if they have risk factors such as comorbidities. Both broadly extended (ST1, ST75) and local lineages (ST1911) were present in our setting.

Agitation down-regulates immunoglobulin binding protein EibG expression in Shiga toxin-producing Escherichia coli (STEC).

Shiga toxin (Stx)-producing Escherichia coli (STEC) carrying eibG synthesize Escherichia coli immunoglobulin binding protein (EibG). EibG nonspecifically binds to immunoglobulins and tends to aggregate in multimers but is poorly expressed in wild-type strains. To study synthesis of the proteins and their regulation in the pathogens, we identified natural growth conditions that increased EibG synthesis. EibG proteins as well as corresponding mRNA were highly expressed under static growth conditions while shearing stress created by agitation during growth repressed protein synthesis. Further regulation effects were driven by reduced oxygen tension, and pH up-regulated EibG expression, but to a lesser extent than growth conditions while decreased temperature down-regulated EibG. Bacteria with increased EibG expression during static growth conditions showed a distinct phenotype with chain formation and biofilm generation, which disappeared with motion. High and low EibG expression was reversible indicating a process with up- and down-regulation of the protein expression. Our findings indicate that shear stress represses EibG expression and might reduce bacterial attachments to cells and surfaces.

Enterohemorrhagic Escherichia coli hemolysin employs outer membrane vesicles to target mitochondria and cause endothelial and epithelial apoptosis.

Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.

Cellular prion proteins in humans and cattle but not sheep are characterized by a low-solubility phenotype.

A feature of transmissible spongiform encephalopathies is the accumulation of infectious prion proteins (PrP(Sc)), which are formed by the conversion of physiological prion proteins (PrP(C)). As PrP(C), which is modified posttranslationally with various types of glycoproteins, serves as the substrates for PrP(Sc) conversion, various PrP(C) subtypes may play a role in the formation of PrP(Sc) and species-specific transmission; the cattle disease BSE is transmissible naturally to humans, but the sheep disease scrapie is not. To reveal new mechanisms modulating prion conversion, we analyzed the PrP(C) profiles by determining the differential PrP(C) protein solubilities in the anionic and nonionic detergents N-lauroylsarcosine, N-octyl-ß-D-glucopyranoside, CHAPS and deoxycholic acid. We compared the resulting solubility profiles of human PrP(C) with the solubility profiles of PrP(C) from sheep and cattle. The PrP(C) subtypes were differentially soluble. However, non-glycosylated PrP(C) from cattle and human was found explicitly in the insoluble fraction, while non-glycosylated ovine PrP(C) was detected in the soluble fraction. These findings indicate the existence of low-solubility PrP(C) phenotypes in cattle and humans.

Effects of metal binding on solubility and resistance of physiological prions depend on tissues and glycotypes.

Prion diseases entail the conversion of a normal host-encoded prion protein (PrP(C)) into an infectious isoform (PrP(Sc)). Various PrP(C) types differing in banding profiles and detergent solubility are present in different tissues, but only few PrP(Sc) types have been generated although PrP(C) acts as substrate. We hypothesize that distinct PrP(C) subtypes may be converted more efficiently to PrP(Sc) than others. One prerequisite for the analysis is the identification of the PrP(C) subtypes present in the protein complexes. Metal binding to PrP(C) is one of the most prominent features of the protein which induces increased proteolysis resistance and structural changes which might play an important role in the conversion process. Here we analyzed the metal-induced structural PrP(C) transformation of two different Triton X-100 soluble PrP(C) types derived from human platelets and brains by changes in protein solubility. We found that zinc and copper rendered approximately half of total PrP(C) and mainly un- and low-glycosylated PrP(C) to the Triton insoluble fraction. Our results indicate the presence of at least two distinct PrP(C) subtypes by metal interactions. The differentiation of high and low soluble metal bound PrP(C) offers precious information about PrP(C) protein composition and provides approaches for analyzing the transformation efficiency to PrP(Sc).

Regional phenotypes of cellular prion proteins in human brains identified by differential detergent solubility.

A hallmark of prion diseases is the accumulation of disease-associated isoforms (PrP(Sc)) which results from the conversion of host-encoded cellular prion proteins (PrP(C)). Using molecular biochemistry, several disease variants of the human Creutzfeldt-Jakob disease have been identified showing several PrP(Sc) variants in individuals and selective accumulation in specific brain regions. As PrP(C) is differentially expressed and post-translationally modified, certain distinct protein compositions may have the ability to convert more efficiently than others. The PrP(C) glycoprotein moiety represents a single yet divers mixture, but little is known about its exact composition. In this study, we separated and characterized PrP(C) derived from six defined human brain regions in regard to their solubility in several detergent solutions and glycoprotein profile formation. We identified four different but regionally distinct protein compositions. PrP(C) found in the neocortex exhibited dominant diglycosylated bands in the high as well as in the low soluble fractions. The proteins in the nucleus lentiformis, thalamus and hippocampus were more soluble with deoxycholic acid as the N-octyl-ß-d-glucopyranoside and the diglycosylated bands displayed strong signals in the supernatants and weaker signals in the sediments. Two different protein profiles were established with PrP(C) derived from the medulla oblongata and the solubility of PrP(C) in the cerebellum clearly differed by the choice of detergent. Our findings indicate the existence of several distinct PrP(C) compositions localized in distinct brain regions. Protein variations may be induced by specific modifications to specific regional biological functions.

Simultaneous detection of three CNS indicator proteins in complex suspensions using a single immuno-PCR protocol.

The diagnosis of infections and protection against their transmission are aided greatly by determination of indicator proteins. However, protein assays are mostly restricted to single-antigen determinations and are often limited in sensitivity and specificity. Consequently, there is a large demand for high-sensitivity immunoassays for analysis of several antigens in protein suspensions. A novel immuno-polymerase chain reaction (PCR) assay is described for the simultaneous detection of central nervous system (CNS) indicators such as the neuron-specific enolase, the glial fibrillary acid protein, and the cellular prion protein. Coated antigens are immunocomplexed with specific antibodies and a DNA fragment is subsequently amplified by PCR. The PCR product obtained corresponds to the antigen signal. Background signals are a critical factor, primarily when using complex protein suspensions, but we were able to reduce background noise dramatically by including two heating steps, the first for protein denaturation and the second for detachment of immunocomplexed DNA, enabling optimal DNA amplification. Using these methods and depending on the antigen and antibody affinity, a sensitivity enhancement of 2 to 3 orders of magnitude is achieved for CNS indicator detection using the immuno-PCR approach compared with ELISA procedures carried out under identical conditions.

Development and applications of simultaneous immunochemical staining and serial detection of overlapping proteins in blotting procedures.

Immunoblotting techniques are widely used for specific protein identifications and characterizations. The specific bindings of antibodies to epitopes in a protein sequence permits determination of antigens and gives detailed information about protein compositions and expression levels in complex suspensions. However the techniques are mostly restricted to one specific antibody determination. Overlaying proteins are detected using numerous repeated gel runs. For multiple but specific protein determinations on one immunoblot, here we describe the detection of several antigens by simultaneous incubation of antibodies originated from different species followed by sequential addition of secondary antibodies labelled with horseradish peroxidase (HRP) and binding to analogous primary antibodies. Particular signals were visualized step by step using a HRP chemiluminescence substrate while enzymatic HRP reactions were meanwhile inactivated irreversibly by hydrogen peroxide incubation. We demonstrate flexible applications of multiple antigen detections using the Western blotting technique with determination of the CNS protein markers neuron specific enolase, glial fibrillary acid protein and the physiological prion protein (PrP(C)) in brains and in meats as food contaminations and with glycotyping of PrP(C) using antibodies binding to different epitopes. We showed the use of the dot blotting technique with serial determination of different antigens in complex protein suspensions. The method is easy to handle and is flexible and applicable in the fields of diagnostics and public health for detection of overlaying proteins on one immunoblot.