Immunoglobulin-binding Proteins Research Articles

Advancements in Immunology and Microbiology Research: A Comprehensive Exploration of Key Areas.

Immunology and microbiology research has witnessed remarkable growth and innovation globally, playing a pivotal role in advancing our understanding of immune mechanisms, disease pathogenesis, and therapeutic interventions. This manuscript presents a comprehensive exploration of the key areas in immunology research, spanning from the utilisation of bacterial proteins as antibody reagents to the intricate realms of clinical immunology and disease management. The utilisation of bacterial immunoglobulin-binding proteins (IBPs), including protein A (SpA), protein G (SpG), and protein L (SpL), has revolutionised serological diagnostics, showing promise in early disease detection and precision medicine. Microbiological studies have shed light on antimicrobial resistance patterns, particularly the emergence of extended-spectrum beta-lactamases (ESBLs), guiding antimicrobial stewardship programmes and informing therapeutic strategies. Clinical immunology research has elucidated the molecular pathways underlying immune-mediated disorders, resulting in tailored management strategies for conditions such as severe combined immunodeficiency (SCID), neuropsychiatric systemic lupus erythematosus (NPSLE), etc. Additionally, significant efforts in vaccine development against tuberculosis and HIV are highlighted, underscoring the ongoing global pursuit of effective preventive measures against these infectious diseases. In summary, immunology and microbiology research have provided significant contributions to global healthcare, fostering collaboration, innovation, and improved patient outcomes.

Establishment of a method to detect human immunoglobulin G1 binding microorganisms

In this study, we developed an original and rapid anti-idiotypic antibody-based ELISA method, different from the techniques defined in the literature for this purpose so far, to detect immunoglobulin binding proteins (IBP) on the surface of bacteria. The test antibody used in our study to detect IBP is a recombinant human immunoglobulin G1 Kappa molecule, and has been used as a drug, Tocilizumab (Actemra®), in humans for therapeutic purposes. As a result, the test antibody in the supernatant after centrifugation is reduced compared to the initial moment due to antibody binding. Staphylococcus aureus cowan 1 strain used as positive control causes at least a 50% decrease in OD value in this respect. A similar observation at this level indicated that among a total of 189 microorganisms tested, 3 Staphylococcus aureus and 1 MRSA carrying high-affinity IgBP showed greater than 50% inhibition. This level of inhibition was not detected in the remaining microorganisms.

Novel Antibody-independent Method to Measure Complement Deposition on Bacteria.

During infection, complement plays a critical role in inflammation, opsonisation, and destruction of microorganisms. This presents a challenge for pathogens such asStaphylococcus aureusto overcome when invading the host. Our current knowledge on the mechanisms that evolved to counteract and disable this system is limited by the molecular tools available. Present techniques utilise labelled complement-specific antibodies to detect deposition upon the bacterial surface, a method not compatible with pathogens such asS. aureus, which are equipped with immunoglobulin-binding proteins, Protein A and Sbi. This protocol uses a novel antibody-independent probe, derived from the C3 binding domain of staphylococcal protein Sbi, in combination with flow cytometry, to quantify complement deposition. Sbi-IV is biotinylated, and deposition is quantified with fluorophore-labelled streptavidin. This novel method allows observation of wild-type cells without the need to disrupt key immune modulating proteins, presenting the opportunity to analyse the complement evasion mechanism used by clinical isolates. Here, we describe a step-by-step protocol for the expression and purification of Sbi-IV protein, quantification and biotinylation of the probe, and finally, optimisation of flow cytometry to detect complement deposition using normal human serum (NHS) and bothLactococcus lactisandS. aureus.

Staphylococcus coagulans possesses many virulence factors of Staph. aureus and Staph. pseudintermedius.

To understand the Staphylococcus coagulans prevalence in causing skin infections in dogs and detection of various virulence genes in Staph. coagulans isolates. Staph. coagulans was isolated from pus swabs collected from dogs with skin infection and identified by detecting thermonuclease, coagulase, and urease genes. The presence of methicillin-resistant gene (mecA) was performed by PCR. Antimicrobial susceptibility test was carried out by disc diffusion method. In total, 38 Staph. coagulans clinical isolates and 42 Staph. coagulans genomes available in NCBI database were screened for 19 virulence genes by PCR and in silico prediction, respectively. A prevalence of 13.8% (38/275) of Staph. coagulans dog skin infection was observed and 15.8% (6/38) of Staph. coagulans isolates carried mecA gene. Many Staph. coagulans isolates were susceptible to all tested antimicrobials. Twenty nine per cent isolates were resistant to ciprofloxacin. Genes encoding leukotoxins, DNase, exfoliative toxin, superantigen-like exotoxin, immunoglobulin-binding proteins, fibrinogen-binding proteins, autolysin, and rod shape-determining protein were detected in almost all the Staph. coagulans clinical isolates and genomes from NCBI database, whereas anti-adhesin plasma-sensitive protein genes were present in relatively lesser number of Staph. coagulans clinical isolates and genomes from NCBI database. Staph. coagulans possesses many virulence factors that are present in other coagulase-positive staphylococci, such as Staph. aureus and Staph. pseudintermedius. The presence of two bi-component leukotoxin genes in tandem with other virulence factor genes in a single pathogenic island in the Staph. coagulans genomes explained their eminence in the virulence of Staph. coagulans causing infections. Staph. coagulans was classified as a separate species in the year 2020 and primarily causes skin infections in dogs. Identification of this species is not included in any of the automated bacterial identification systems. Hence, many veterinary laboratories do not have a strategy to identify this bacterium. This study will help in the identification of Staph. coagulans in veterinary laboratories by PCR apart from detecting various virulence factors present in this pathogen. The existence of many virulence factors and prevalence in different animals in varied geographical locations suggest that Staph. coagulans is an important coagulase-positive staphylococcal pathogen in animals.

ATR-FTIR Biosensors for Antibody Detection and Analysis.

Quality control of drug products is of paramount importance in the pharmaceutical world. It ensures product safety, efficiency, and consistency. In the case of complex biomolecules such as therapeutic proteins, small variations in bioprocess parameters can induce substantial variations in terms of structure, impacting the drug product quality. Conditions for obtaining highly reproducible grafting of 11-mercaptoundecanoic acid were determined. On that basis, we developed an easy-to-use, cost effective, and timesaving biosensor based on ATR-FTIR spectroscopy able to detect immunoglobulins during their production. A germanium crystal, used as an internal reflection element (IRE) for FTIR spectroscopy, was covalently coated with immunoglobulin-binding proteins. This thereby functionalized surface could bind only immunoglobulins present in complex media such as culture media or biopharmaceutical products. The potential subsequent analysis of their structure by ATR-FTIR spectroscopy makes this biosensor a powerful tool to monitor the production of biotherapeutics and assess important critical quality attributes (CQAs) such as high-order structure and aggregation level.

Novel method for detecting complement C3 deposition on Staphylococcus aureus

The primary host response to Staphylococcus aureus infection occurs via complement. Complement is an elegant evolutionarily conserved system, playing essential roles in early defences by working in concert with immune cells to survey, label and destroy microbial intruders and coordinate inflammation. Currently the exact mechanisms employed by S. aureus to manipulate and evade complement is not clear and is hindered by the lack of accurate molecular tools that can report on complement deposition on the bacterial surface. Current gold-standard detection methods employ labelled complement-specific antibodies and flow cytometry to determine complement deposited on bacteria. These methods are restricted by virtue of the expression of the S. aureus immunoglobulin binding proteins, Protein A and Sbi. In this study we describe the use of a novel antibody-independent C3 probe derived from the staphylococcal Sbi protein, specifically Sbi-IV domain. Here we show that biotin-labelled Sbi-IV interacts specifically with deposited C3 products on the staphylococcal surface and thus can be used to measure complement fixation on wild-type cells expressing a full repertoire of immune evasion proteins. Lastly, our data indicates that genetically diverse S. aureus strains restrict complement to different degrees suggesting that complement evasion is a variable virulence trait among S. aureus isolates.

Highly Multiplexed Serology for Nonhuman Mammals.

ABSTRACTEmerging infectious diseases represent a serious and ongoing threat to humans. Most emerging viruses are maintained in stable relationships with other species of animals, and their emergence within the human population results from cross-species transmission. Therefore, if we want to be prepared for the next emerging virus, we need to broadly characterize the diversity and ecology of viruses currently infecting other animals (i.e., the animal virosphere). High-throughput metagenomic sequencing has accelerated the pace of virus discovery. However, molecular assays can detect only active infections and only if virus is present within the sampled fluid or tissue at the time of collection. In contrast, serological assays measure long-lived antibody responses to infections, which can be detected within the blood, regardless of the infected tissues. Therefore, serological assays can provide a complementary approach for understanding the circulation of viruses, and while serological assays have historically been limited in scope, recent advancements allow thousands to hundreds of thousands of antigens to be assessed simultaneously using <1 μL of blood (i.e., highly multiplexed serology). The application of highly multiplexed serology for the characterization of the animal virosphere is dependent on the availability of reagents that can be used to capture or label antibodies of interest. Here, we evaluate the utility of commercial immunoglobulin-binding proteins (protein A and protein G) to enable highly multiplexed serology in 25 species of nonhuman mammals, and we describe a competitive fluorescence-linked immunosorbent assay (FLISA) that can be used as an initial screen for choosing the most appropriate capture protein for a given host species.IMPORTANCE Antibodies are generated in response to infections with viruses and other pathogens, and they help protect against future exposures. Mature antibodies are long lived, are highly specific, and can bind to their protein targets with high affinity. Thus, antibodies can also provide information about an individual’s history of viral exposures, which has important applications for understanding the epidemiology and etiology of disease. In recent years, there have been large advances in the available methods for broadly characterizing antibody-binding profiles, but thus far, these have been utilized primarily with human samples only. Here, we demonstrate that commercial antibody-binding reagents can facilitate modern antibody assays for a wide variety of mammalian species, and we describe an inexpensive and fast approach for choosing the best reagent for each animal species. By studying antibody-binding profiles in captive and wild animals, we can better understand the distribution and prevalence of viruses that could spill over into humans.

Universal Enzyme-Linked Immunosorbent Assays (ELISA) and Utility in the Detection of Antibodies against Salmonella spp. in Several Animal Species

The aim of this study was to confirm the feasibility of using hybrid immunoglobulin-binding reagents in ELISAs for IgG/IgY detection and detection of specific antibodies against an infectious microorganism (Salmonella spp.) in various animal species using a universal diagnostic ELISA. Hybrid immunoglobulin-binding bacterial proteins (IBP), including recombinant protein LA, recombinant protein LG, and recombinant protein AG, have been produced to improve their binding affinity to a much larger number of immunoglobulins. Thus, this hybrid bacterial protein represents a powerful tool for the binding, detection, and purification of immunoglobulins and their fragments. However, SpLA-LG-peroxidase and SpLAG-anti-IgY-peroxidase were produced using the periodate method. These compounds have been shown to be effective as reagents. Their binding affinity to immunoglobulins surpasses that of previously reported hybrid IgG-binding proteins, including the most known SpAG, SpLA, and SpLG. The IgY fraction was isolated from the egg yolks of various birds, including chicken, bantam hen, guinea hen, quail, goose, duck, wild and domestic pigeons, parakeets, cattle egrets, pheasants, and ostrich. The IgY fraction was isolated using the chloroform-polyethylene glycol (PEG) method. An ELISA for anti-Salmonella spp. antibodies was employed with some modifications to determine the presence of antibodies in humans, laying hens, geese, quails, and pigeons. Salmonella is a motile, flagellated, rod-shaped zoonotic pathogen that can survive in the presence or absence of oxygen. They belong to the family Enterobacteriaceae and are implicated in typhoid fever and food-borne illnesses. This pathogen is associated with several diseases, which may become fatal and negatively impact the health of individuals and various economies globally. The poultry industry is most vulnerable to the influence of this pernicious microbe. The authors concluded that universal enzyme-linked immunosorbent assays were effective and reproducible in detecting immunoglobulins from both avian and mammalian species, but ELISA using the SpLAG-anti-IgY-HRP conjugate only reacted with the whole panel of animal antibodies. This conjugate was further used to standardize a universal ELISA for the determination of anti-Salmonella antibodies, in which human and avian species were serologically assessed.

Silent Antibodies Start Talking: Enhanced Lateral Flow Serodiagnosis with Two-Stage Incorporation of Labels into Immune Complexes.

The presence of pathogen-specific antibodies in the blood is widely controlled by a serodiagnostic technique based on the lateral flow immunoassay (LFIA). However, its common one-stage format with an antigen immobilized in the binding zone of a test strip and a nanodispersed label conjugated with immunoglobulin-binding proteins is associated with risks of very low analytical signals. In this study, the first stage of the immunochromatographic serodiagnosis was carried out in its traditional format using a conjugate of gold nanoparticles with staphylococcal immunoglobulin-binding protein A and an antigen immobilized on a working membrane. At the second stage, a labeled immunoglobulin-binding protein was added, which enhanced the coloration of the bound immune complexes. The use of two separated steps, binding of specific antibodies, and further coloration of the formed complexes, allowed for a significant reduction of the influence of non-specific immunoglobulins on the assay results. The proposed approach was applied for the serodiagnosis using a recombinant RBD protein of SARS-CoV-2. As a result, an increase in the intensity of test zone coloration by more than two orders of magnitude was demonstrated, which enabled the significant reduction of false-negative results. The diagnostic sensitivity of the LFIA was 62.5% for the common format and 100% for the enhanced format. Moreover, the diagnostic specificity of both variants was 100%.

Discovery of Novel Circulating Immune Complexes in Lupus Nephritis Using Immunoproteomics.

ObjectiveThe goal is to discover novel circulating immune complexes (ICx) in the serum of lupus nephritis (LN) as potential biomarkers.MethodsProtein A/G magnetic beads or C1q-coated plates were used to capture ICx in the serum of LN, followed by the identification of immunoglobulin-binding proteins using liquid chromatography and tandem mass spectrometry (LC-MS/MS). Bioinformatic approaches and single-cell RNA sequencing (scRNA Seq) databases were used to select potential candidate ICx markers in LN. The selected ICx markers were further validated using ELISA.ResultsA total of 300 immunoglobulin-binding proteins were discovered in the screening, among which 77 proteins were detectable only in LN samples. Bioinformatics-assisted selection allowed us to further identify 10 potential immunoglobulin-binding proteins, which form ICx as potential biomarkers in LN. In a validation cohort of 62 LN patients and 21 healthy controls (HC), we found that prolyl 3-hydroxylase 1 (P3H1), phosphatase and actin regulator 4 (PHACTR4), and regulator of G-protein signaling 12 (RGS12) ICx exhibited discriminative capability in distinguishing LN from HC, with an area under the curve (AUC) values of 0.82, 0.99, and 0.90, respectively. Furthermore, a biomarker panel comprising CD14, CD34, cystatin A, myocyte enhancer factor 2C (MEF2C), RGS12, and ubiquitin C (UBC) ICx could distinguish active LN from inactive LN with an AUC value of 0.85, which is comparable to or better than pathological parameters such as renal activity index (AI) and renal chronicity index (CI).ConclusionImmunoproteomics-based discovery studies have enabled us to identify circulating immune complexes as potential biomarkers of LN.

Comparative secretome analysis of Staphylococcus aureus strains with different within-herd intramammary infection prevalence

ABSTRACT Staphylococcus aureus is a major pathogen causing intramammary infection and mastitis in dairy cows. S. aureus genotypes (GT) can differ significantly in their ability to diffuse and persist in the herd; while the association of virulence gene carriage with epidemiological behavior remains unclear, a role for secreted proteins has been postulated. We characterized the secretome of six S. aureus strains belonging to two genotypes with opposite within-herd prevalence, GTB (high) and GTS (low), corresponding to sequence types (ST) 8 and 398, by high-resolution tandem mass spectrometry and differential analysis with Proteome Discoverer. Data are available via ProteomeXchange with identifier PXD029571. Out of 720 identified proteins, 98 were unique or more abundant in GTB/ST8 and 68 in GTS/ST398. GTB/ST8 released more immunoglobulin-binding proteins, complement and antimicrobial peptide inhibitors, enterotoxins, and metabolic enzymes, while GTS/ST398 released more leukocidins, hemolysins, lipases, and peptidases. Furthermore, GTB/ST8 released the von Willebrand factor protein, staphylokinase, and clumping factor B, while GTS released the staphylococcal coagulase and clumping factor A. Hence, GTB/ST8 secretomes indicated a higher propensity for immune evasion and chronicity and GTS/ST398 secretomes for cellular damage and inflammation, consistent with their epidemiological characteristics. Accordingly, GTS/ST398 secretions were significantly more cytotoxic against bovine PBMCs in vitro. Our findings confirm the crucial role of extracellular virulence factors in S. aureus pathogenesis and highlight the need to investigate their differential release adding to gene carriage for a better understanding of the relationship of S. aureus genotypes with epidemiological behavior and, possibly, disease severity.

MBOVPG45_0375 Encodes an IgG-Binding Protein and MBOVPG45_0376 Encodes an IgG-Cleaving Protein in Mycoplasma bovis.

Mycoplasma bovis is a significant bacterial pathogen which is able to persist in cattle and cause chronic diseases. This phenomenon may relate to M. bovis evading the immune system of the host. Immunoglobulin-binding proteins are widely distributed in a variety of pathogenic bacteria, including some Mycoplasma species. These proteins are considered to help the bacteria evade the immune response of the host. Here we found M. bovis strain PG45 can bind to IgG from several animals. MBOVPG45_0375 encodes a putative membrane protein, has strong amino acid sequence similarity with Immunoglobulin G-binding protein in Mycoplasma mycoides subsp. capri. Hence, we constructed recombinant MBOVPG45_0375 (r0375) in the Escherichia coli expression system and demonstrated that r0375 can bind to IgG non-immunologically rather than specific binding similar to interaction of antigen and antibody. Moreover, r0375 can bind to the Fab fragment of IgG. Also, the binding of r0375 and IgG inhibits the formation of antigen-antibody union. Furthermore, MBOVPG45_0376 encodes an IgG-cleaving protein of M. bovis strain PG45. Nevertheless, r0375 binding to IgG is required for the cleavage activity of recombinant 0376 (r0376). The activity of r0376 is also affected by incubation time and temperature. In addition, we found both MBOVPG45_0375 and MBOVPG45_0376 are membrane proteins of M. bovis strain PG45. These results about MBOVPG45_0375 as an IgG-binding protein and MBOVPG45_0376 as an IgG-cleaving protein offer a new insight into the interaction between M. bovis and its host.

Identification of Novel Proteins in Foam Nests of the Japanese Forest Green Tree Frog, Rhacophorus arboreus.

Foam nests of frogs are natural biosurfactants that contain potential compounds for biocompatible materials, Drug Delivery System (DDS), emulsifiers, and bioremediation. To elucidate the protein components in the foam nests of Rhacophorus arboreus, which is an endemic Japanese frog species commonly seen during the rainy season, we performed amino acid analysis, SDS-PAGE electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry using intact foam nests. Many proteins were detected in these foam nests, ranging from a few to several hundred kDa, with both essential and non-essential amino acids. Next, we performed transcriptome analysis using a next-generation sequencer on total RNAs extracted from oviducts before egg-laying. The soluble foam nests were purified by LC-MS and analyzed using Edman degradation, and the identified N-terminal sequences were matched to the transcriptome data. Four proteins that shared significant sequence homologies with extracellular superoxide dismutase of Nanorana parkeri, vitelline membrane outer layer protein 1 homolog of Xenopus tropicalis, ranasmurfin of Polypedates leucomystax, and alpha-1-antichymotrypsin of Sorex araneus were identified. Prior to purification of the foam nests, they were treated with both a reducing reagent and an alkylating agent, and LC-MS/ MS analyses were performed. We identified 22 proteins in the foam nests that were homologous with proteinase inhibitors, ribonuclease, glycoproteins, antimicrobial protein and barrier, immunoglobulin-binding proteins, glycoprotein binding protein, colored protein, and keratin-associated protein. The presence of these proteins in foam nests, along with small molecules, such as carbohydrates and sugars, would protect them against microbial and parasitic attack, oxidative stress, and a shortage of moisture.

Throughput optimized immuno‐infrared‐assay for Alzheimer’s disease diagnosis

AbstractBackgroundIt is believed, that one of the first events in the progression of Alzheimer’s Disease is the misfolding of the Amyloid Beta (Aβ) Peptide. This event takes place up to 20 years before the clinical onset of the disease. Therefore, biosensors detecting the misfolding of Aβ are especially useful for the early and specific detection of Alzheimer’s Disease. In recent studies, our biosensor based on ATR‐FTIR spectroscopy performed with an overall accuracy of 86% [1,2,3]. This system is robust and reliable but the user input required is very high because each sensor can only be used once. To overcome these issues and increase the automation capacity, a regenerative system is urgently needed.MethodTo achieve higher throughput of this system, we introduced immunoglobulin binding proteins (Protein A and Protein G) as capture for antibody immobilization. Protein A and Protein G are able to recognize and bind the constant region of different IgG antibodies in a non‐covlant manner. This allows multiple measurements within the same system by performing multiple binding and elution cycles.ResultProtein A and Protein G can be attached covalently to the sensor without losing its ability to bind IgG antibodies [4]. The antibody binding and elution is monitored by ATR‐FTIR spectroscopy and validated using fluorescence spectroscopy. It is possible to perform at least 35 cycles of antibody binding and elution over 7 days without loss in diagnostic accuracy. The sensor is still able to extract different biomarkers like Aβ and the Tau Protein from CSF with highly comparable results compared to the recently used system.ConclusionThe development of a regenerative immuno‐infrared‐sensor is crucial to reduce the user input and to increase the throughput of this method. Compared to assays where the capture antibody is covalently immobilized, the regenerative system has a factor 6 increased thoughput.

Genome-wide identification and characterization of Solanum tuberosum BiP genes reveal the role of the promoter architecture in BiP gene diversity

The endoplasmic reticulum (ER) immunoglobulin binding proteins (BiPs) are molecular chaperones involved in normal protein maturation and refolding malformed proteins through the unfolded protein response (UPR). Plant BiPs belong to a multi-gene family contributing to development, immunity, and responses to environmental stresses. This study identified three BiP homologs in the Solanum tuberosum (potato) genome using phylogenetic, amino acid sequence, 3-D protein modeling, and gene structure analysis. These analyses revealed that StBiP1 and StBiP2 grouped with AtBiP2, whereas StBiP3 grouped with AtBiP3. While the protein sequences and folding structures are highly similar, these StBiPs are distinguishable by their expression patterns in different tissues and in response to environmental stressors such as treatment with heat, chemicals, or virus elicitors of UPR. Ab initio promoter analysis revealed that potato and Arabidopsis BiP1 and BiP2 promoters were highly enriched with cis-regulatory elements (CREs) linked to developmental processes, whereas BiP3 promoters were enriched with stress related CREs. The frequency and linear distribution of these CREs produced two phylogenetic branches that further resolve the groups identified through gene phylogeny and exon/intron phase analysis. These data reveal that the CRE architecture of BiP promoters potentially define their spatio-temporal expression patterns under developmental and stress related cues.

Detecting total immunoglobulins in diverse animal species with a novel split enzymatic assay

BackgroundTotal immunolobulin G concentration is a useful, albeit underutilized, diagnostic parameter for health assessments of non-domestic animal species, due to a lack of functional diagnostic tools. Traditional assays, including enzyme-linked immunosorbent assay or radial immunodiffusion, require development of specific reagents (e.g., polyclonal antisera and appropriate protocols) for each animal species, precluding wide and easy adoption in wildlife welfare. As an alternative, bacterial virulence factors able to bind IgGs in antigen-independent manner can be used. To further simplify the diagnostic procedure and increase the number of species recognized by an assay, in this study a recently developed Split Trehalase immunoglobulin assay (STIGA) with bIBPs as a sensing elements was used to detect antibodies in 29 species from 9 orders. Three bacterial immunoglobulin binding proteins (protein G, protein A and protein L) were incorporated into STIGA reagents to increase the number of species recognized.ResultsIgG concentrations were detected through glucose production and produced signals were categorized in 4 categories, from not active to strong signal. Activation was detected in almost all tested animal species, apart from birds. Incorporation of Protein G, Protein A and Protein L allowed detection of IgGs in 62, 15.5 and 6.9% of species with a strong signal, respectively. Assays combining 2 bacterial immunoglobulin binding proteins as sensing element generally gave poorer performance than assays with the same bacterial immunoglobulin binding proteins fused to both trehalase fragments.ConclusionsSTIGA assays have potential to be further developed into an easily adoptable diagnostic test for total amount of IgGs in almost any serum sample, independent of species.

Reversible Immuno-Infrared Sensor for the Detection of Alzheimer's Disease Related Biomarkers.

The development of biosensors for medical purposes is a growing field. An immuno-infrared biosensor for the preclinical detection of Alzheimer's disease (AD) in body fluids was developed. The key element of this sensor is an ATR crystal with chemically modified surface to catch the biomarker out of the body fluid. So far, the immuno-infrared sensor can be used only once and requires time-consuming steps of sensor exchange, sensor cleaning, and novel surface functionalization. Here, we developed an immuno-infrared sensor providing a reusable surface and showcase its performance by the detection of the AD biomarker proteins Aβ and Tau in human cerebrospinal fluid (CSF). The sensor surface is covalently coated with the immunoglobulin binding proteins Protein A or Protein G. These were employed for noncovalent immobilization of antibodies and the subsequent immobilization and analysis of their antigens. The reversible antibody immobilization can be repeated several times with the same or different antibodies. Further, the more specific binding of the antibody via its Fc region instead of the conventional NHS coupling leads to a 3-4-fold higher antigen binding capacity of the antibody. Thus, the throughput, sensitivity, and automation capacity of the immuno-infrared biosensor are significantly increased as compared to former immuno-infrared assays. This immuno-sensor can be used with any antibody that binds to Protein A or Protein G.

An Acid Up-Regulated Surface Protein of Lactobacillus paracasei Strain GCRL 46 is Phylogenetically Related to the Secreted Glucan- (GpbB) and Immunoglobulin-Binding (SibA) Protein of Pathogenic Streptococci.

Bacterial cell wall hydrolases, including amidases and peptidases, play a critical role in peptidoglycan turnover during growth, impacting daughter cell separation, and cell death, through autolysis. When exploring the regulation of protein expression across the growth cycle of an acid-resistant strain of Lactobacillus paracasei, GCRL 46, we observed temporal up-regulation of proteins in the 40–45 kDa molecular weight range for whole-cell extracts when culturing in fermenters at a controlled pH of 4.0 versus optimum growth pH of 6.3. Up-regulation of proteins in this size range was not detected in SDS-PAGE gels of the cytosolic fraction, but was routinely detected following growth at low pH in whole cells and cell debris obtained after bead beating and centrifugation, indicating a cell surface location. N-terminal sequencing and in silico analyses showed sequence similarity with proteins in the L. casei group (L. casei, L. paracasei and L. rhamnosus) which were variously annotated as uncharacterized proteins, surface antigens, possible TrsG proteins, CHAP (cysteine, histidine-dependent amidohydrolases/peptidases)-domain proteins or putative peptidoglycan d,l-endopeptidase due to the presence of a CwlO domain. This protein is a homologue of the p40 (Msp2) secreted protein of L. rhamnosus LGG, which is linked to probiotic functionality in this species, and is phylogenetically related to structurally-similar proteins found in Enterococcus, Streptococcus and Bifidobacterium species, including the glucan-binding (GbpB), surface antigen (SagA) proteins detected in pathogenic group A streptococci species as secreted, immunoglobulin-binding (SibA) proteins (also named PcsB). Three-dimensional (3D) modelling predicted structural similarities in the CHAP proteins from the L. casei group and streptococcal species, indicating retention of overall architecture despite sequence divergence, and an implied retention of function during evolution. A phylogenetically-related hydrolase also contained the CwlO domain with a NLPC_P60 domain, and showed similar overall but distinct architecture to the CHAP proteins. We concluded that the surface-located, CHAP protein in L. casei is up-regulated during long-term exposure to acidic conditions during growth but not during acid shock.

IgY antibodies of chicken do not bind staphylococcal binder of immunoglobulin (Sbi) from Staphylococcus aureus

The immunoglobulin (Ig) binding proteins of Staphylococcus aureus namely staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulin (Sbi) are responsible for false positives during immunoassays. Avian IgY antibodies were reported to have no affinity to SpA and thus are safe for use in immunoassays. However, the behaviour of Sbi with IgY was not reported. The purpose of the present study is to evaluate the interactions between IgY antibodies and Sbi protein from different S. aureus strains. Initially, heterologous cloning and expression of complete sbi gene in Escherichia coli was undertaken. Recombinant Sbi protein was utilized to generate polyclonal anti-Sbi IgY and anti-Sbi antibodies in chicken and BALB/c mice respectively. Indirect ELISA and Western blotting were performed to evaluate the reactivity of anti-Sbi antibodies. Non-reducing PAGE followed by Western blotting and double-antibody sandwich dot-ELISA were performed to analyze the reactivity of IgY antibodies with recombinant Sbi and native Sbi from S. aureus strains. To avoid the possible interference of enzyme-conjugated secondary antibodies from mammalian sources, mouse anti-Sbi revealing antibodies were labeled with biotin so that streptavidin-HRP was used as developing reagent for chromogenic reaction. Sbi was highly immunogenic in chicken and mouse with antibody titers of 1:128,000 and 1:64,000 dilutions respectively. We observed that unimmunized IgY antibodies showed no affinity to either recombinant Sbi or native Sbi from S. aureus strains in Western blotting and double antibody sandwich ELISA. In view of these observations, we recommend that IgY antibodies are safe and free from false positives due to SpA and Sbi in immunoassays involving detection of S. aureus antigens/exotoxins.

Protein A and Protein G Purification of Antibodies.

Protein A and Protein G are immunoglobulin-binding proteins expressed in Staphylococcus aureus and Streptococcus sp., respectively, that have been adapted for use in purifying large amounts of IgG. They are available covalently attached to affinity resins such as 4% cross-linked agarose, making them suitable for low-pressure antibody isolation. Protein A is not recommended for the isolation of mouse mAbs because it lacks affinity for mouse IgG1, or for the isolation of antibodies from sheep, goat, chicken, hamster, or rat. IgGs from most species bind to Protein G at near physiological pH and ionic strength with a higher affinity than IgG binding to Protein A. Therefore, the pH required to dissociate bound IgG is lower, resulting in the loss of activity for some antibodies. If this is observed, Protein A may be an alternative if the IgG from the species being isolated can be purified using Protein A. Neither Protein A nor Protein G can be used for the isolation of chicken antibodies.