The molecular characterization of antibody binding to a superantigen-like protein from a commensal microbe.

Microorganisms have coevolved diverse mechanisms to impair host defenses. A major one, superantigens, can result in devastating effects on the immune system. While all known superantigens induce vast immune cell proliferation and come from opportunistic pathogens, recently, proteins with similar broad specificity to antibody variable (V) domain families were identified in a commensal microbiota. These proteins, identified in the human commensal Ruminococcus gnavus, are called immunoglobulin-binding protein (Ibp) A and B and have been shown to activate B cells in vitro expressing either human VH3 or murine VH5/6/7. Here, we provide molecular and functional studies revealing the basis of this Ibp/immunoglobulin (Ig) interaction. The crystal structure and biochemical assays of a truncated IbpA construct in complex with mouse VH5 antigen-binding fragment (Fab) shows a binding of Ig heavy chain framework residues to the Ibp Domain D and the C-terminal heavy chain binding domain (HCBD). We used targeted mutagenesis of contact residues and affinity measurements and performed studies of the Fab-IbpA complex to determine the stoichiometry between Ibp and VH domains, suggesting Ibp may serve to cluster full-length IgA antibodies in vivo. Furthermore, in vitro stimulation experiments indicate that binding of the Ibp HCBD alone is sufficient to activate responsive murine B cell receptors. The presence of these proteins in a commensal microbe suggest that binding a broad repertoire of immunoglobulins, particularly in the gut/microbiome environment, may provide an important function in the maintenance of host/microbiome homeostasis contrasting with the pathogenic role of structurally homologous superantigens expressed by pathogens.

Validity of immunoglobulin-binding protein 1 as a biomarker for lupus nephritis.

BACKGROUND: Lupus nephritis (LN) is a major issue that adds a burden on patients with systemic lupus erythematosus (SLE). Immunoglobulin-binding protein 1 (IGBP1) is identified as a phosphoprotein that has been recently reported to be linked to the B-cell receptor complex and regulates differentiation, proliferation, apoptosis, and tolerance of B cells. Its diagnostic and/or prognostic role in LN has been highlighted only recently. OBJECTIVES: This study aims to evaluate the relation between serum IGBP1 and SLE disease activity and/or renal activity and to investigate the validity of IGBP1 as a biomarker for LN. METHODS: 96 participants were enrolled and divided into three groups: nephritis, nonnephritis, and control groups. The patients with SLE were diagnosed according to the Systemic Lupus International Collaborating Clinics classification (SLICC) criteria. The serum IGBP1 level was assayed using an enzyme-linked immunosorbent assay (ELISA). Assessments were conducted using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2k) and renal biopsy for LN patients. RESULTS: The nephritis and nonnephritis groups had higher IGBP1 levels than the controls; the nephritis group had the highest serum IGBP1 levels (p < .001). Significant correlations were found between IGBP1 levels and proteinuria (r = 0.568, p = .001) and renal SLEDAI (r = 0.475, p = .006) in the nephritis group; on the other hand, the correlation of serum IGBP1 levels with SLEDAI-2K was non-significant for both groups (nephritis and nonnephritis groups). The IGBP1 levels were significantly different among histopathologic classes (p < .001), with class V showing the highest level. Moreover, it showed a significant positive correlation with the pathologic activity index. Compared with renal SLEDAI for identifying active renal affection in patients with SLE, the serum IGBP1 level with a cut-off value of 547.45 ng/mL is a valid biomarker for detecting active nephritis with 93.8% sensitivity and 96.9% specificity. CONCLUSION: The serum IGBP1 levels were high in patients with LN and were positively correlated with the pathologic activity index. The serum IGBP1 level of 547.45 ng/mL is a valid biomarker for detecting active nephritis. Thus, we recommend that clinicians monitor the serum IGBP1 level of patients with SLE to detect LN.

Staphylococcus aureus second immunoglobulin-binding protein drives atopic dermatitis via IL-33.

BACKGROUND: Staphylococcus aureus is the dominant infective trigger of atopic dermatitis (AD). How this bacterium drives type 2 allergic pathology in the absence of infection in patients with AD is unclear. OBJECTIVE: We sought to identify the S aureus-derived virulence factor(s) that initiates the cutaneous type 2-promoting immune response responsible for AD. METHODS: In vitro human keratinocyte cell culture, ex vivo human skin organ explants, and the eczema-prone Nishiki-nezumi Cinnamon/Tokyo University of Agriculture and Technology strain mouse were used as model systems to assess type 2-promoting immune responses to S aureus. Identification of the bioactive factor was accomplished using fast protein liquid chromatography and mass spectrometry. Bioactivity was confirmed by cloning and expression in an Escherichia coli vector system, and S aureus second immunoglobulin-binding protein (Sbi) mutant strains confirming loss of activity. RESULTS: S aureus was unique among staphylococcal species in its ability to induce the rapid release of constitutive IL-33 from human keratinocytes independent of the Toll-like receptor pathway. Using the eczema-prone Nishiki-nezumi Cinnamon/Tokyo University of Agriculture and Technology strain mouse model, we showed that IL-33 was essential for inducing the immune response to S aureus in vivo. By fractionation and candidate testing, we identified Sbi as the predominant staphylococcus-derived virulence factor that directly drives IL-33 release from human keratinocytes. Immunohistology of skin demonstrated that corneodesmosin, a component of corneodesmosomes that form key intercellular adhesive structures in the stratum corneum, was disrupted, resulting in reduction of skin barrier function. CONCLUSIONS: S aureus-derived Sbi is a unique type 2-promoting virulence factor capable of initiating the type 2-promoting cytokine activity underlying AD.

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.

Plasma immunoglobulin binding protein 1 as a predictor of development of lupus nephritis.

Objective: Urine levels of immunoglobulin binding protein 1 (IGBP1) are increased in patients with lupus nephritis (LN) compared with systemic lupus erythematosus (SLE) patients without nephritis. However, the clinical significance of IGBP1 level in plasma is unclear. We aimed to evaluate whether the plasma level of IGBP1 can predict future development of LN in SLE patients without nephritis. Methods: Forty-three SLE patients without nephritis were followed for 5 years. Plasma IGBP1 levels were measured using ELISA, and clinical and laboratory data were obtained at study entry. Development of LN was confirmed by renal biopsy. Cox regression analysis was performed to identify factors associated with development of LN, and receiver operating characteristic curve analysis was used to determine the predictive value of each factor. Results: Of the total 43 patients, eight (18.6%) developed LN during the follow-up period. Compared with patients who did not develop LN, those who developed LN had higher levels of plasma IGBP1 (6.3 ng/ml (range 4.3­9.6 ng/mL) vs. 13.3 ng/ml (range 7.2­31.3 ng/ml); p=0.023). In the Cox regression analysis, higher CRP (hazard ratio (HR)=1.325, 95% confidence interval (CI) 1.073­1.637, p=0.009), anti-dsDNA antibody (Ab; HR=1.066, 95% CI 1.012­1.124, p=0.017) and plasma IGBP1 (HR=1.091, 95% CI 1.034­1.152, p=0.002) were associated with future development of LN. Among these factors, anti-dsDNA Ab (area under the curve (AUC)=0.893) had the highest predictive value followed by plasma IGBP1 (AUC=0.761) and CRP (AUC=0.634). A combination of anti-dsDNA Ab and plasma IGBP1 as a composite predictor was highly specific (97%) for predicting the development of LN. Conclusions: Plasma IGBP1 can be used complementarily with anti-dsDNA Ab for detecting SLE patients at a higher risk of developing LN.

Immunoglobulin Binding Protein 1 as a Potential Urine Biomarker in Patients with Lupus Nephritis.

We evaluated the role of immunoglobulin binding protein 1 (IGBP1), a phosphoprotein associated with the B cell receptor (BCR) complex, as a urine biomarker in lupus nephritis (LN). The IGBP1 concentrations in plasma and urine of patients with LN, systemic lupus erythematosus (SLE) without nephritis and healthy controls were estimated by ELISA. IGBP1 expression in the kidneys of LN patients and transplantation donors was detected by immunohistochemistry. Microarray-based global gene expression profile of HK-2 cells with IGBP1 knock-down and fluorescence-activated cell sorting (FACS) for intracellular IGBP1 expression in human peripheral blood mononuclear cells (PBMCs) was performed. Urine IGBP1 levels were elevated significantly in LN patients, and it correlated with the clinical activity indices (complement 3 (C3) level, anti-dsDNA antibodies titer, SLE Disease Activity Index-2000 (SLEDAI-2K) and histological activity index. IGBP1 expression was increased in LN patients as compared to the donors and was detected mainly in the tubules by histopathology. In microarray analysis, several genes related to SLE pathogenesis (PPME1, ROCK2, VTCN1, IL-17R, NEU1, HLA-DM, and PTX3) responded to siRNA-mediated IGBP1 silencing. In FACS, IGBP1 was expressed mainly in the CD14+ cells. The overall expression of IGBP1 in PBMCs was higher in LN patients as compared with that in SLE patients without nephritis. Conclusively, urinary IGBP1 may be a novel biomarker reflecting the clinical and histological activities in LN.

Staphylococcus aureus Regulatory RNAs as Potential Biomarkers for Bloodstream Infections.

Staphylococcus aureus is a commensal bacterium and pathogen. Identifying biomarkers for the transition from colonization to disease caused by this organism would be useful. Several S. aureus small RNAs (sRNAs) regulate virulence. We investigated presence and expression of 8 sRNAs in 83 S. aureus strains from 42 patients with sepsis or septic shock and 41 asymptomatic colonized carriers. Small pathogenicity island sRNAs sprB and sprC were clade specific. Six sRNAs had variable expression not correlated with clinical status. Expression of RNAIII was lower in strains from septic shock patients than in strains from colonized patients. When RNAIII was associated with expression of sprD, colonizing strains could be discriminated from strains in patients with bloodstream infections, including patients with sepsis and septic shock. Isolates associated with colonization might have sRNAs with target expression different from those of disease isolates. Monitoring expression of RNAIII and sprD could help determine severity of bloodstream infections.

Protein Biosynthesis and Maturation in the ER.

The endoplasmic reticulum takes care of the folding, assembly, and quality control of thousands of proteins destined to the different compartments of the endomembrane system or to be secreted in the apoplast. Here we describe how these early events in the life of all these proteins can be followed biochemically by using velocity or isopycnic ultracentrifugation, metabolic labelling with radioactive amino acids, drug treatments, and immunoselection in various conditions and, in certain cases, predicted in silico by algorithms.

The Protective Role of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Protein Sigma-1 Receptor in Regulating Endothelial Inflammation and Permeability Associated with Acute Lung Injury.

Earlier studies from our lab identified endoplasmic reticulum (ER) chaperone BiP/GRP78, an important component of MAM, to be a novel determinant of endothelial cell (EC) dysfunction associated with acute lung injury (ALI). Sigma1R (Sig1R) is another unique ER receptor chaperone that has been identified to associate with BiP/GRP78 at the MAM and is known to be a pluripotent modulator of cellular homeostasis. However, it is unclear if Sig1R also plays a role in regulating the EC inflammation and permeability associated with ALI. Our data using human pulmonary artery endothelial cells (HPAECs) showed that siRNA-mediated knockdown of Sig1R potentiated LPS-induced the expression of proinflammatory molecules ICAM-1, VCAM-1 and IL-8. Consistent with this, Sig1R agonist, PRE-084, known to activate Sig1R by inducing its dissociation from BiP/GRP78, blunted the above response. Notably, PRE-084 failed to blunt LPS-induced inflammatory responses in Sig1R-depleted cells, confirming that the effect of PRE-084 is driven by Sig1R. Furthermore, Sig1R antagonist, NE-100, known to inactivate Sig1R by blocking its dissociation from BiP/GRP78, failed to block LPS-induced inflammatory responses, establishing that dissociation from BiP/GRP78 is required for Sig1R to exert its anti-inflammatory action. Unlike Sig1R, the siRNA-mediated knockdown or Subtilase AB-mediated inactivation of BiP/GRP78 protected against LPS-induced EC inflammation. Interestingly, the protective effect of BiP/GRP78 knockdown or inactivation was abolished in cells that were depleted of Sig1R, confirming that BiP/GRP78 knockdown/inactivation-mediated suppression of EC inflammation is mediated via Sig1R. In view of these findings, we determined the in vivo relevance of Sig1R in a mouse model of sepsis-induced ALI. The intraperitoneal injection of PRE-084 mitigated sepsis-induced ALI, as evidenced by a decrease in ICAM-1, IL-6 levels, lung PMN infiltration, and lung vascular leakage. Together, these data evidence a protective role of Sig1R against endothelial dysfunction associated with ALI and identify it as a viable target in terms of controlling ALI in sepsis.

Study of ER stress and apoptotic proteins in the heart and tumor exposed to doxorubicin.

Although a high cumulative dose of Doxorubicin (Dox) is known to cause cardiotoxicity, there is still a lack of understanding of the subcellular basis of this drug-induced cardiomyopathy. Differential effects of Dox on mitochondria and endoplasmic reticulum (ER) were examined in cardiomyocytes, tumor cells, implanted tumors and hearts of normal as well as tumor-bearing animals. Dox increased mitochondrial (Mito) Bax activation at 3 h in the cardiomyocyte without change in the DNA damage inducible transcriptor-3 (DDIT3) expression in the ER. Increased DDIT3 in these Dox-treated cardiomyocytes at 24 h suggested that increased MitoBax may have promoted ER stress related changes in DDIT3. Dissociation of immunoglobulin-binding protein (Bip) from activating transcription factor 6 (ATF6)-Bip complex in the ER was observed as an adaptive response to Dox. In contrast, breast cancer MCF7 cells showed an ER stress response to Dox with increased DDIT3 as early as 3 h which may have triggered a positive feedback activation of ATF6 at 12 and 24 h and promoted Calnexin. At these later time points, increased Bax activation in cancer cells suggested that MitoBax may be controlled by DDIT3 or by Calnexin. DDIT3 response in tumors was evoked by Dox, however this response was inversely correlated with increased Bip and Bax expression in hearts from tumor bearing animals. It is suggested that in Dox-induced cardiotoxicity both mitochondrial and ER stresses play an integral role through a mutual interaction where an inhibition of DDIT3 or Calnexin may also be crucial to achieve Dox resistance in cardiomyocytes.

Development of multi-epitope vaccine constructs for non-small cell lung cancer (NSCLC) against USA human leukocyte antigen background: an immunoinformatic approach toward future vaccine designing.

OBJECTIVES: The design of peptide-based vaccines for cancer is a promising immunotherapy that can induce a cancer-specific cytotoxic response in tumor cells. METHODS: Herein, we used the immunoinformatic approach in designing a multi-epitope vaccine targeting G-protein coupled receptor 87 (GPCR-87), cystine/glutamate transporter (SLC7A11), Immunoglobulin binding protein 1 (IGBP1), and thioredoxin domain-containing protein 5 (TXNDC5), which can potentially contribute to NSCLC. The MHC-I and MHC-II epitopes selected for the fusion construct were evaluated for their antigenic and non-allergenic natures via VaxiJen and AllerTop. RESULTS: A total of five epitopes, four class-I (FIFYLKNIV, CRYTSVLFY, RYLKVVKPF, and RQAKIQRYK), and one class-II (NQVRGYPTLLWFRDG), having combined USA population coverage of 100%, were used to make ten possible multi-epitope fusion constructs. In these constructs, PADRE, a universal T-helper epitope, and RSO9, a TLR4 agonist, were fused as adjuvants. The molecular docking analysis revealed that two constructs were showing significant binding affinities toward HLA-A*02:01, the most prevalent HLA allele in USA. Moreover, MD simulations marked one construct as a promising therapeutic candidate. CONCLUSION: The multi-epitope vaccine constructs designed using immunogenic, and non-allergenic peptides of NSCLS tumor-associated proteins are likely to pose significant therapeutic efficacies in cancer immunotherapy due to their high binding affinities toward HLA molecules.

Histophilus somni: Antigenic and Genomic Changes Relevant to Bovine Respiratory Disease.

Histophilus somni is associated with several disease syndromes in cattle and plays an important role in the bovine respiratory disease complex. H somni isolates exhibit significant differences in terms of susceptibility to inactivation by normal serum corresponding to the general ability to cause clinical disease. Isolates possess a variety of virulence factors, and variation in virulence factor expression is well recognized and associated with antigenic differences. Sequencing of genes associated with known virulence factors has identified genetic variability between isolates. The antigenic and genomic differences represent significant challenges to the host immune system and are problematic for vaccine design.

ATPase activity of human binding immunoglobulin protein (BiP) variants is enhanced by signal sequence and physiological concentrations of Mn2.

B-cell immunoglobulin binding protein (BiP) is an essential endoplasmic reticulum (ER) chaperone normally found in the ER lumen. However, BiP also has other extracellular and intracellular functions. As it is unclear whether peripheral BiP has a signal and/or ER retention sequence, here we produced and biochemically characterised four variants of BiP. The variants differed depending on the presence or the absence of signal and ER retention peptides. Proteins were purified using nickel affinity chromatography, and variant size and quality were confirmed using SDS/PAGE gels. The thermal denaturing temperature of these proteins was found to be 46-47 °C. In addition, we characterised nucleotide binding properties in the absence and the presence of divalent cations. Interestingly, in the absence of cations, ADP has a higher binding affinity to BiP than ATP. The presence of divalent cations results in a decrease of the Kd values of both ADP and ATP, indicating higher affinities of both nucleotides for BiP. ATPase assays were carried out to study the enzyme activity of these variants and to characterise the kinetic parameters of BiP variants. Variants with the signal sequence had higher specific activities than those without. Both Mg2+ and Mn2+ efficiently stimulated the ATPase activity of these variants at low micromolar concentrations, whereas calcium failed to stimulate BiP ATPase. Our novel findings indicate the potential functionality of BiP variants that retain a signal sequence, and also reveal the effect of physiological concentrations of cations on the nucleotide binding properties and enzyme activities of all variants.

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.

A mode of error: Immunoglobulin binding protein (a subset of anti-citrullinated proteins) can cause false positive tuberculosis test results in rheumatoid arthritis.

Citrullinated Immunoglobulin Binding Protein (BiP) is a newly described autoimmune target in rheumatoid arthritis (RA), one of many cyclic citrullinated peptides(CCP or ACPA). BiP is over-expressed in RA patients causing T cell expansion and increased interferon levels during incubation for the QuantiFERON-Gold tuberculosis test (QFT-G TB). The QFT-G TB has never been validated where interferon is increased by underlying disease, as for example RA. Of ACPA-positive RA patients (n = 126), we found a 13% false-positive TB test rate by QFT-G TB. Despite subsequent biologic therapy for 3 years of all 126 RA patients, none showed evidence of TB without INH. Most of the false-positive RA patients after treatment with biologic therapy reverted to a negative QFT-G test. False TB tests correlated with ACPA level (p < 0.02). Three healthy women without arthritis or TB exposure had negative QFT-G TB. In vitro, all three tested positive every time for TB correlating to the dose of BiP or anti-BiP added, at 2 ug/ml, 5 ug/ml, 10 ug/ml, and 20 ug/ml. BiP naturally found in the majority of ACPA-positive RA patients can result in a false positive QFT-G TB. Subsequent undertreatment of RA, if biologic therapy is withheld, and overtreatment of presumed latent TB may harm patients.

BiP3 supports the early stages of female gametogenesis in the absence of BiP1 and BiP2 in Arabidopsis thaliana.

Immunoglobulin binding protein (BiP) is an essential heat shock protein 70 (Hsp70) in the endoplasmic reticulum (ER) that functions in various processes including protein translocation, protein folding and quality control. Arabidopsis thaliana harbors ubiquitously expressed genes BIP1 and BIP2, as well as BIP3, which is induced only by ER stress. Recently, we reported that these BIP genes are expressed in male gametophytes and cooperate with each other to support male gametogenesis and pollen competitiveness. Here, we report that the BIP genes cooperate to support female gametogenesis. As reported previously, the bip1 bip2 double mutation causes defects in the fusion of polar nuclei during female gametogenesis. By contrast, the bip triple mutant female gametophytes exhibited defects during the early stages of female gametophyte development, which suggests that BIP3 supports the early stages of female gametophyte development, but not polar nuclear fusion, in the absence of BiP1 and BiP2.

Effect of Duhuo Jisheng Tang on PERK/Bip Signaling Pathway in Knee Osteoarthritis Model Rats / 中国实验方剂学杂志

Objective: To explore the possible mechanism of Duhuo Jisheng Tang in relieving knee osteoarthritis based on protein kinase R-like endoplasmic reticulum kinase (PERK)/immunoglobulin-binding protein (Bip) signaling pathway.Method: A model of knee osteoarthritis was established by cold stimulation.Rats were randomly divided into blank group,model group,celecoxib group (0.021 g·kg-1),low,medium and high-dose Duhuo Jisheng Tang groups (8.37,16.72,33.48 g·kg-1).Blank group and model group were given equal volume of physiological saline.The changes of knee joint diameter were recorded.The pathological changes of rat articular cartilage were observed by hematoxylin-eosin (HE) staining.The expressions of tumor necrosis factor-alpha (TNF-α),interleukin-1β(IL-1β) and hyaluronic acid (HA) in serum were detected by enzyme-linked immunosorbent assay (ELISA).The mRNA and protein expression levels of PERK,Bip and cysteinyl as parates pecific protein-9(Caspase-9) in cartilage were detected by Real-time PCR and Western blot.Result: The knee joint redn ess and the joint diameter of celecoxib group and high-dose Duhuo Jisheng Tang group were improved,and the joint diameter was reduced significantly (Pα,IL-1β and HA were increased in model group (PPPα,IL-1β and HA in serum of celecoxib group and high-dose Duhuo Jisheng Tang group were decreased (PPPPConclusion: Duhuo Jisheng Tang can alleviate the symptoms of knee osteoarthritis model rats,and its mechanism may be related to the regulation of PERK/Bip signaling pathway in rat cartilage.

Applications for an engineered Protein-G variant with a pH controllable affinity to antibody fragments.

Immunoglobulin binding proteins (IBPs) are broadly used as reagents for the purification and detection of antibodies. Among the IBPs, the most widely used are Protein-A and Protein-G. The C2 domain of Protein-G from Streptococcus is a multi-specific protein domain; it possesses a high affinity (K(D) ~10 nM) for the Fc region of the IgG, but a much lower affinity (KD~low µM) for the constant domain of the antibody fragment (Fab), which limits some of its applications. Here, we describe the engineering of the Protein-G interface using phage display to create Protein-G-A1, a variant with 8 point mutations and an approximately 100-fold improved affinity over the parent domain for the 4D5 Fab scaffold. Protein-G-A1 is capable of robust binding to Fab fragments for numerous applications. Furthermore, we isolated a variant with pH-dependent affinity, demonstrating a 1,000-fold change in affinity from pH7 to 4. Additional rational mutagenesis endowed Protein-G with significantly enhanced stability in basic conditions relative to the parent domain while maintaining high affinity to the Fab. This property is particularly useful to regenerate Protein-G affinity columns. Lastly, the affinity-matured Protein-G-A1 variant was tethered together to produce dimers capable of providing multivalent affinity enhancement to a low affinity antibody fragment-antigen interaction. Engineered Protein-G variants should find widespread application in the use of Fab-based affinity reagents.

Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species / Comparación de las técnicas de detección de la reactividad de la proteína de unión de la inmunoglobulina frente a la inmunoglobulina producida por diferentes especies aviarias y mamíferas

The rationale of this study was to use several immunological assays to investigate the reactivity of immunoglobulin binding protein (IBP) to immunoglobulins from various avian and mammalian species. The IBP studied were Staphylococcal protein A (SpA), Streptococcal protein G (SpG), Peptostreptococcal protein L (SpL) and recombinant protein LA (SpLA). The various immunological techniques used were double immunodiffusion (Ouchterlony technique) that tested positive high protein reactivities, direct and competitive enzyme-linked immunosorbent assays (ELISAs) that tested moderate and low positive protein binding capacities, respectively. In addition to sandwich ELISAs, immunoblot analyses and Ig-purification by SpA-affinity chromatography, which were sensitive tests and helpful in the screening and confirmatory tests were also used. The Ouchterlony technique showed that compared to the other proteins, SpLA had the highest range of reactivity with animal sera and purified immunoglobulins while SpL was least reactive. With the direct ELISA, SpL reacted with the raccoon sera, rabbit IgG and with IgY from bantam hens and pigeons. While with the direct ELISA, SpA reacted with sera from skunk, coyote, raccoon, mule, donkey and human. The sandwich ELISA revealed high reactivity of both SpG and SpLA with mammalian sera titres ranging from 1:32 (raccoon serum) to 1:1024 (mule and donkey sera).These results suggest that IBP can be used for the detection of immunoglobulin using various immunological assays and this is important for the diagnosis of infectious diseases in animal and bird populations studied and in the purification of immunoglobulins.

El fundamento de este estudio radica en el uso de varios ensayos inmunológicos para investigar la reactividad de la proteína de unión de la inmunoglobulina (IBP) frente a las inmunoglobulinas de varias especies aviarias y mamíferas. Las proteínas IBP estudiadas fueron la proteína estafilocócica A (SpA), la proteína estreptocócica G (SpG), la proteína peptoestreptocócica L (SpL), y la proteína recombinante LA (SpLA). Las varias técnicas inmunológicas usadas fueron: la inmunodifusión doble (técnica de Ouchterlony) para examinar las reactividades positivas de la proteína alta; el ensayo por inmunoabsorción ligado a enzimas(ELISA), de tipo directo y competitivo, para examinar la capacidad de realizar uniones positivas de proteína moderada y baja, respectivamente, además del ensayo ELISA 'Sándwich', los análisis inmunoblot, yla purificación de IgG, mediante cromatografía de afinidad, los cuales fueron pruebas sensibles y útiles en el tamizaje y las pruebas de confirmación. La técnica de Ouchterlony mostró que - en comparación con otras proteínas - la SpLA tenía el grado más alto de reactividad con los sueros animales y las inmunoglobulinas purificadas, mientras que la SpL fue la menos reactiva. Con el ELISA directo, la SpL reaccionó con los sueros de mapache, la IgG de conejo, así como con la IgY de palomas y gallinas de Bantam, en tanto con el ELISA directo, la SpA reaccionó con sueros de mofeta, coyote, mapache, mula, asno y seres humanos. ELISA "sándwich" reveló una alta reactividad tanto de SpG como de SpLA, con títulos séricos mamíferos que iban desde 1:32 (suero de mapache) hasta 1:1024 (sueros de mula y de asno). Estos resultados sugieren que la proteína de unión IBP puede usarse en la detección de la inmunoglobulina usando varios ensayos inmunológicos, lo cual es importante para el diagnóstico de enfermedades infecciosas en las poblaciones animales y aviarias bajo estudio, así como para la purificación de inmunoglobulinas.